18 Staphylococci, Streptococci, Meningococci, Gonococci

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    Chair of Microbiology, Virology, and Immunology

    Pathogenic cocci

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    Classification. Staphylococci are included in theFirmicutes 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.

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    Morphology. Staphylococci are spherical inshape, 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.

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    Main

    characteristics

    S. aureus S. epider-

    midis

    S. sapro-

    phyticus

    Plasmacoagulase +

    Phosphatase + +

    Reductase + +

    Protein A, super-

    ficial antigen

    +

    Mannitol + +

    Trehalose + +

    Production ofalpha-toxin

    +

    Resistance to

    novobiocin

    S S R

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    Virulence factors

    Staphylococciexpress 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 givenfactor. This also reflects the inadequacies of many

    animal models for staphylococcal diseases.

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    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 isactivated 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.

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    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 thebacteria, 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.

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    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.

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    Treatment. Staphylococcal diseases are treated

    with antibiotics (penicillin, phenoxymethylpenicillin,

    tetracycline, gramicidin, etc.), sulphonamides

    (norsulphazol, sulphazol, etc.), and antistaphylococcalgamma-globulin.

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    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).

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    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 alwaysconstantly), and break down salicin and trehalose, with acid

    formation.

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    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.

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    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.

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    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 milkin 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 andto penicillin (a number of strainsshow growth in media

    containing 0.5-1 U of antibiotic per 1 ml of media). All

    enterococci decarboxylate tyrosine.

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    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 theE. coli.In lesions of

    the duodenum, gall bladder, and urinary tract enterococci arefound as a result of dysbacteriosis. Isolation of enterococci

    serves as a criterion of contamination of water, sewage, and

    foodstuffs with faeces.

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    Streptococcus pneumoniae

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    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 enterthe 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 thepharyngeal 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.

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    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.

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    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.

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    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 inlarge 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 titreof antibodies, namely streptolysins O and antihyaluronidase.

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    Meningococci

    The meningococcus (Neisseria meningitidis) was

    isolated from the cerebrospinal fluid of patientswith meningitis and studied in detail in 1887 by A.

    Weichselbaum. At present the organism is

    classified in the genus Neisseria, family

    Neisseriaceae

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    Cultivation. Optimum temperature for growth is36-37 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 toinoculation as the organism is extremely susceptible to

    temperatures above or below 37 degrees.

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    Fermentative properties. Meningococci do notliquefy 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. meningitidisis its antiphagocytic polysaccharide

    capsule. Fimbriae are factor of virulence

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    Antigenic structure and classification.Meningococci were found

    to contain three fractions: carbohydrate (C) which is common to allmeningococci, 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.

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    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.

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    Laboratory diagnosis.Specimens of cerebrospinal fluid,

    nasopharyngeal discharge, blood, andorgans obtained at autopsy are used for

    examination.

    The following methods of investigation

    are employed: (1) microscopic

    examination of cerebrospinal fluidprecipitate; (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.

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    GonococciThe causative agent of gonorrhoea and

    blennorrhoea (Neisseria gonorrhoeae) was

    discovered in 1879 by A. Neisser in suppurative

    discharges. In 1885 E. Bumm isolated a pureculture of the organism and studied it in detail.

    Gonococci belong to the genus Neisseria, family

    Neisseriaceae.

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    Fermentative properties. The gonococcus

    possesses low biochemical activity and noproteolytic 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. gonorrhoeaethat may play a role in virulence

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    Designation Location Contribution

    PileMajor fimbrial

    protein

    Initial binding to epithelial cells

    P.I (Por) Outer membraneporin

    May prevent phagolysosome

    formation in neutrophils and/or

    reduce oxidative burst

    LOS Outer membranelipooligosaccharideElicits inflammatory response,triggers release of TNF

    P.III (Rmp) Outer membraneprotein

    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

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    The WHO expert committee has

    recommended listing the gonococcal infection

    among infectious diseases with compulsoryregistration 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 ofuniform criteria of clinical and laboratory

    diagnosis, and treatment of gonococcal infection

    and more efficient methods for determining the

    sensitivity of circulating gonococci to variousdrugs are also recommended by the committee.