Transcript LOS

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 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.
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 IgG 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: 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,
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




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.


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
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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
The
Catalase test determines if the organism produces the
enzyme “Catalase”, which breaks down hydrogen peroxide
(H2O2) to water and oxygen (O2).
Catalase
2 H 2 O2
Catalase
2 H 2O
+
O2
(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
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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 pH.
If a pH indicator is present in the medium (such as Phenol
red) a color change will occur dependant upon the pH of
the medium (agar or broth).
Mannitol Salt Agar contains the pH indicator “Phenol Red”
This pH indicator is red at neutral pH (around 7.0), but
turns yellow under acidic conditions.
Antibiotic Susceptibility/Resistance
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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 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

Bubbling indicates a positive test
for the presence of the catalase
enzyme.
Coagulase

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.

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

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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 endproduct that changes the red pH
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


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, sulphazol, 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 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.
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); groupspecific 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. 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 betahaemolytic 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.
Laboratory diagnosis is made on the basis of
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 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.
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 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.
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.
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, 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.
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 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 (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 OmpA 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
IgA1 proteases, which cleave the heavy chain of the human
immunoglobulin at different points within the hinge region. Split
products of IgA1 have been found in the genital secretions of
women with gonorrhea, suggesting that the neisserial IgA1
protease is present and active during genital infection. It is
thought that the Fab fragments of IgA1 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 that
block bactercidal antibodies
against P.I and LOS
Tbp1
Tbp2
Lbp
binding
to
epithelial
and Outer membrane
Iron acquisition for growth
Outer membrane
receptor for
Iron acquisition for growth
receptors for
transferrin
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.