Chair of Microbiology, Virology, and Immunology

Pathogenic cocci

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 (Gforms) 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- S. sapromidis

phyticus

Plasmacoagulase

Phosphatase

Reductase

Protein A, superficial antigen

Mannitol

Trehalose

Production of
alpha-toxin

Resistance to
novobiocin

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 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;
(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 betahaemolytic 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
Major fimbrial
Pile

Contribution
Initial binding to epithelial cells

protein

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

and 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.