CHAIR OF MEDICAL BIOLOGY,
MICROBIOLOGY, VIROLOGY AND
IMMUNOLOGY
CORYNEBACTERIA.
LISTERIA
Prof. S. Klymnyuk
Classification. The genus Corynebacterium comprises a
species pathogenic for human beings and several species
which are non-pathogenic for man and conditionally
designated as diphtheroids. The majority of diphtheroids
occurs in the external environment (water, soil, air), some
of them are present as commensals in the human body.
Japanese scientists isolated Corynebacterium kusaya from
brines used in cavalla canning; it does not form volutin
granules. Its presence in brine prevents spoiling of fish
products during salting and drying.
Species
Pathogenicity for humans and animals
C. diphtherias
Pathogenic for humans, causes diphtheria
C.
pseudotuberculosis
Pathogenic for sheep, goats, horses, and other
warm-blooded animals, sometimes causes in
fection in humans
Non-pathogenic for humans, dwells on eye
mucosa
Induces pyelitis and cystitis in experimental
animals and pyelonephritis in calves
C. xerosis
C. renale
C. kulschen
Parasitizes in the body of mice and rats
C. pseudodiphtheriae
Non-pathogenic for humans, dwells on the
mucous membrane of the nasopharynx
C. equi
Detected in pneumonia in animals, weakly
pathogenic for experimental animals
C. bovis
Causes mastitis in animals, found in milk
Causative Agent of Diphtheria. Extensive clinical,
pathoanatomical, epidemiological, and experimental
investigations preceded the discovery of the agent
responsible for diphtheria. They paved the way for the
discovery of the organism (E.Klebs, 1883), its isolation in
pure culture (F. Loeffler, 1884), separation of the toxin (E.
Roux and A. Yersin, 1888), antitoxin (E. Behring and
S.Kitasato, 1890) and diphtheria toxoid (G. Ramon, 1923).
Morphology. Corynebacterium diphtheriae (L. coryna
club) is a straight or slightly curved rod, 1-8 mcm in length
and 0.3-0.8 mcm in breadth. The organism is pleomorphous
and stains more intensely at its ends, which contain volutin
granules (Babes-Ernst granules, metachromatin). C.
diphtheriae frequently display terminal club-shaped
swellings. Branched forms as well as short, almost coccal,
forms sometimes occur. In smears the organisms are
arranged at an angle and resemble spread-out fingers. They
are Gram-positive and produce no spores, capsules, or
flagella.
Corynebacterium diphtheriae, Gram’s
technique
Corynebacterium diphtheriae, Neisser’s
technique
Corynebacterium diphtheriae,
Loeffler’s technique
Corynebacterium diphtheriae,
Loeffler’s technique
Corynebacteria of the gravis
biovar produce large, rough
(R-forms), rosette-like black
or grey colonies on tellurite
agar
which
contains
defibrinated
blood
and
potassium tellurite. The
organisms ferment dextrin,
starch, and glycogen and
produce a pellicle and a
granular deposit in meat
broth. They are usually
highly toxic with very
marked invasive properties.
Biovar gravis
The colonies produced by
corynebacteria of the mitis
biovar on tellurite agar are dark,
smooth (S-forms), and shining.
Starch and glycogen are not
fermented,
and
dextrin
fermentation is not a constant
property. The organisms cause
haemolysis of all animal
erythrocytes and produce diffuse
turbidity in meat broth. Cultures
of this biovar are usually less
toxic and invasive than those of
the gravis biovar.
Biovar mitis
Organisms
of
the
intermedius
biovar
are
intermediate strains. They
produce small (RS-forms)
black colonies on tellurite
agar. Starch and glycogen
are not fermented. Growth in
meat
broth
produces
turbidity and a granular
deposit.
Biovar intermedius
Fermentative properties. All three biovars of C.
diphtheriae do not coagulate milk, do not break down urea,
produce no indole, and slowly produce hydrogen sulphide.
They reduce nitrates to nitrites. Potassium tellurite is also
reduced, and for this reason C. diphtheriae colonies grown
on tellurite agar turn black or grey. Glucose and levulose
are fermented whereas galactose, maltose, starch, dextrin,
and glycerin fermentation is variable. Exposure to factors
in the external environment renders the organisms
incapable of carbohydrate fermentation.
Biochemical Properties of Corynebacterium Species
Strain
Me- ProducFermentation of
tation of
chr
om Ca- Py- Gel Ure Lac Ma Tre Sta Glu
atic ta- razi atin ase to- lto- hal rch cose
se ose
se
Gra lase na- ase
mid
nul
ase
es
C. diphtheriae
Var. mitis
Var. gravis
Var intermedius
+
+
+
+
+
+
–
–
–
–
–
–
–
–
–
–
–
–
+
+
+
–
–
–
–
+
–
+
+
+
C. ulcerans
+
+
–
+
+
–
+
+
+
+
C. pseudotuberculosis
+
+
–
–
+
–
+
–
–
+
C. pseudodiphtheriticum
+
+
+
–
+
–
–
–
–
–
C. xerosis
+
+
+
–
–
–
–
–
–
+
Antigenic structure. Eleven serovars of C. diphtheriae
have been deter-mined on the basis of the agglutination
reaction. They all produce toxins which do not differ from
each other and are neutralized completely by the standard
diphtheria antitoxin. A number of authors have confirmed
the presence of type-specific thermolabile surface protein
antigens (K-antigens) and group-specific thermostable
somatic polysaccharide antigens (O-antigens) in the
diphtheria corynebacteria
Toxin production. In broth cultures C. diphtheriae
produce potent exotoxins (histotoxin, dermonecrotoxin,
haemolysin). The toxigenicity of these organisms is linked
with lysogeny (the presence of moderate phages-prophages
in the toxigenic strains). The classical International
standard strain, Park-Williams 8 exotoxin-producing strain,
is also lysogenic and has retained the property of toxin
production for over 85 years. The genetic determinants of
toxigenicity (tox+ genes) are located in the genome of the
prophage, which is integrated with the C. diphtheriae
nucleoid.
The diphtheria exotoxin is a complex of more than 20
antigens. It has been obtained in a crystalline form.
C. diphtheriae also contain bacteriocines (corynecines)
which provide these organisms with certain selective
advantages.
The diphtheria toxin contains large amounts of aminonitrogen and catalyses chemical reaction in the body. The
toxigenic strains of C. diphtheriae are characterized by
marked dehydrogenase activity, while the non-toxigenic
strains do not possess such activity.
Diphtheria toxin is excreted from the bacterium as a single
polypeptide chain of about 61,000 daltons with two
disulfide bridges. Although highly toxic for cells or
animals, the pure, intact toxin is inert in cell-free protein
systems, even when NAD is present. Thus, the secreted
toxin is actually a proenzyme which, in cell-free systems,
must be activated before it can function as an enzyme. This
activation is accomplished in two steps: (1) treatment with
trypsm hydrolyzes a peptide bond between the disulfidelinked amino acids; and (2) reduction of the disulfides to
sulfhydryl groups using a reducing agent such as
mercaptoethanol yields two smaller peptides, which have
been designated fragment A (21,150 daltons) and fragment
B (40,000 daltons).
Fragment A is active in cleaving the nicotinamide moiety from NAD
and in catalyzing the transfer of ADP-ribose from NAD to EF-2
when added to cell-free, protein-synthesizing systems, but it has no
effect when given to animals or to intact HeLa cells. Thus, although
fragment A is the activated enzyme (and hence contains all the toxic
properties), it cannot get into intact cells.
Fragment B, on the other hand, has no enzymatic activity, but it is
needed for attachment of the toxin tospecific receptor sites on cells.
Cells possess specific glycoprotein receptor sites for the diphtheria
toxin, as suggested by the following observation: Rats and mice are
over 1000 times more resistant to the intact toxin than are other
susceptible animals, but their cell-free protein-synthesizing system is
equally sensitive to the enzymatic action of fragment A. Moreover,
toxin that is defective in its A fragment (and is, therefore, nontoxic)
but retains a normal B fragment, will competitively inhibit the action
of normal toxin on HeLa cells.
In summary, the usual series of events leading to toxin
action is as follows: (1) the toxin binds to specific receptor
sites on susceptible cells; (2) the toxin enters the cell
(perhaps through a phagocytic vesicle that can then fuse
with a lysosome), and lysosomal proteases hydrolyze the
toxin into fragments A and B; and (3) reduction of the
disulfide bridges (perhaps by glutathione) releases
fragment A from fragment B; and (4) fragment A can then
enzymatically inactivate EF-2.
The diphtheria toxin is unstable, and is destroyed easily by
exposure to heat, light, and oxygen of the air, but is
relatively resistant to super-sonic vibrations. The toxin is
transformed into the toxoid by mixture with 0.3-0.4 per
cent formalin and maintenance at 38-40° C for a period of
3 or 4 weeks. The toxoid is more resistant to physical and
chemical factors than the toxin.
Place for bindig with cell’s
recerptor
Toxin’s structure
Bacteriophage with tox+ gene
Pathogenicity for animals. Animals do not naturally
acquire diphtheria. Although, virulent diphtheria
organisms were found to be pre-sent in horses, cows, and
dogs, the epidemiological significance of animals in
diphtheria is negligible.
Among the laboratory animals, guinea pigs and rabbits are
most susceptible to the disease. Inoculation of these
animals with a culture or toxin gives rise to typical
manifestations of a toxinfection and the appearance of
inflammation, oedema, and necrosis at the site of
inoculation. The internal organs become congested,
particularly the adrenals in which haemorrhages occur.
Toxic reactions in animal
Pathogenesis
Pathogenesis and disease in man. Patients suffering from
the disease and carriers are the sources of infection in
diphtheria. The disease is transmitted by an air-droplet
route, and sometimes with dust particles. Transmission by
various objects (toys, dishes, books, towels, handkerchiefs,
etc.) and foodstuffs (milk, cold dishes, etc.) contaminated
with C. diphtheriae is also possible.
Histotoxin plays the principal role in the pathogenesis of
diphtheria. It blocks protein synthesis in the cells of
mammals and inactivates transferase, the enzyme
responsible for the formation of the polypeptide chain.
C. diphtheriae penetrate into the blood and tissues of sick
humans and infected animals. The diffusion factor due to
which these organisms are capable of invasion is formed of
a complex of K-antigen and lipids of the wall of bacterial
cells. The lipids contain corynemicolic and corynemicolenic
acids, the cord factor (trehalose dimicolate), and mannose
and inositol phosphatides. The cord factor causes the death
of mice, destroys mitochondria, and disturbs the processes
of respiration and phosphorylation. The necrotic factor,
alpha-glutaric acid, and haemolysin are considered to be
factors of invasiveness.
Clinical studies and experiments on animals have provided
evidence of the influence of pathogenic staphylococci and
streptococci, on the development of diphtheria, the
infection becoming more severe in the presence of these
organisms. Hypersensitivity to C. diphtheriae and to the
products of their metabolism is of definite significance in
the pathogenesis of diphtheria.
In man, membranes containing a large number of C. diphtheriae and
other bacteria are formed at the site of entry of the causative agent
(pharynx, nose, trachea, eye conjunctiva, skin, vulva, vagina, and
wounds). The toxin produces diphtheria! inflammation and necrosis in
the mucous membranes or skin. On being absorbed, the toxin affects the
nerve cells, cardiac muscle, and parenchymatous organs and causes
severe toxaemia.
Deep changes take place in the cardiac muscle, vessels, adrenals, and
in the central and peripheral nervous systems.
According to the site of the lesion, faucial diphtheria and diphtheritic
croup occur most frequently, and nasal diphtheria somewhat less
frequently. The incidence of diphtheria of the eyes, ears, genital organs,
and skin is relatively rare. Faucial diphtheria constitutes more than 90
per cent of all the diphtherial cases, and nasal diphtheria takes the second
place.
Immunity following diphtheria depends mainly on the antitoxin con-tent
m the blood However, a definite role of the antibacterial component,
associated with phagocytosis and the presence of opsonins, agglutinins,
precipitins, and complement-fixing substances cannot be ruled out.
Therefore, immunity produced by diphtheria is anti-infectious (anti-toxic
and antibacterial) in character.
Schick test. This test is used for detecting the presence of antitoxin in
children's blood. The toxin is injected intracutaneously into the forearm
in a 0 2 ml volume which is equivalent to 1/40 DLM for guinea pigs. A
positive reaction, which indicates susceptibility to the disease, is
manifested by an erythematous swelling measuring 2 cm in diameter
which appears at the site of injection in 24-48 hours. The Schick test is
positive when the blood contains either no antitoxin or not more
than0.005 units per millilitre of blood serum. A negative Schick reaction
indicates, to a certain degree, insusceptibility to diphtheria.
In view of the fact that the diphtheria exotoxin produces a
state of sensitization and causes the development of severe
reaction in many children, it is advisable to restrict the
application of the Schick test and conduct it with great
care.
Children from 1 to 4 years old are most susceptible to
diphtheria. A relative increase of the incidence of the
disease among individuals 15years of age and older has
been noted in recent years.
Diphtheria leaves a less stable immunity than do other
children's diseases (measles, whooping cough). Diphtheria
reinfection occurs in 6-7 per cent of the cases.
Laboratory diagnosis. Discharges from the pharynx, nose,
and, some-times, from the vulva, eyes, and skin are collected
with a sterile cotton-wool swab for examination.
The toxigenic and non-toxigenic strains of diphtheria
corynebacteria are differentiated either by subcutaneous
or intracutaneous infection of guinea pigs, or by the agar
precipitation method, the latter being relatively simple
and may be carried out in any laboratory. It is based on
the ability of the diphtheria toxin to react with the antitoxin
and produce a precipitate resembling arrow-tendrils.
Necrotic angina
Treatment. According to the physician's prescriptions,
patients are given antitoxin in doses ranging from 5000 to
15000 units in mildly severe cases, and from 30 000 to 50
000 units in severe cases of the disease. Penicillin,
streptomycin, tetracycline, erythromycin, sulphonamides,
and cardiac drugs are also employed. Diphtheria toxoid is
recommended in definite doses for improving the
immunobiological state of the body, i.e for stimulating
antitoxin production.
Prophylaxis. General control measures comprise early
diagnosis, prompt hospitalization, thorough disinfection of
premises and objects, recognition of carriers, and
systematic health education.
Specific prophylaxis is afforded by active immunization. A
number of preparations are used: the pertussis-diphtheria
vaccine, purified adsorbed toxoid, pertussis-diphtheriatetanus vaccine All preparations are used according to
instructions and directions.