Gram Positive Cocci

Two Genera
Staphylococcus

Streptococcus

are nonmotile
do not form spores

They are distinguished by two main criteria

1. Microscopically :
staphylococci appear in grapelike clusters
streptococci are in chain

2. Biochemically:
staphylococci produce catalase an important virulence
factor (they degrade hydrogen peroxid- H2 O2 that is
microbicidal in O2 , H2O )
streptococci do not
 STREPTOCOCCUS
Structure
Streptococci are cocci that occur in pairs or chains :
Gram-positive
nonmotile
nonsporeforming
catalase-negative.
older cultures may lose their Gram-positive character.
some are facultative anaerobes, and some are obligate (strict) anaerobes
most require enriched media (blood agar)

Group A streptococci have a hyaluronic acid capsule

 CLASSIFICATION

CLINICAL
Pyogenic Streptococci
Oral Streptococci
Enteric Streptococci
Peptostreptococci grow under aerobic or microaerophilic
conditions and produce variable hemolysis ) P. magnus , P.
anaerobius

HEMOLYSYS
alpha-hemolysis (incomplete, green hemolysis),
beta-hemolysis (clear, complete lysis of red cells)
gamma-hemolysis(no hemolysis).
SEROLOGICAL-Lancefield (A-H), (K-U)
is based on antigenic differences in cell wall carbohydrates
(groups A to V), in cell wall pili-associated protein, and in the
polysaccharide capsule in group B streptococci.
BIOCHEMICAL (physiological)
Important antigens of beta hemolytic streptococci :
C carbohydrate group specific antigen
determines the group of hemolytic streptococci
is located in the cell wall ,
its specificity is determined by an amino sugar ( gr A ; group specific carbohydrate is
a dimer of N-acetylglucosamine and rhamnose )
M , T proteins type specific antigens
M
is the most important virulence factor ,
- it consists of two polypeptide chains complexed in an alpha helix
determines the type of group A hemolytic streptococci
there are approximately 100 serotypes based on the M protein
- are subdivided into class I and class II molecules
- the class I M proteins share exposed antigens, whereas the class II M proteins do not
have exposed shared antigens
antibody to M protein provides type specific immunity
is the main antiphagocytic component of S. pyogenes (also has a polysaccharide capsule
that plays a role in retarding phagocitosis)
T(trypsin-resistant) protein
SPECIES LANCEFIELD TYPICAL HEMOLYSIS
GROUP

S. pyogenes A Beta

S. agalactiae B Beta

E. faecalis D Alpha or beta or none

S. bovis D Alpha or none

S. pneumoniae NA Alpha

Viridans group NA Alpha

 GROUP A

S. pyogenes
opportunistic pathogen is responsable for about
90% of all cases of pharyngitis
a common form of pharyngitis is "Strep throat" -
is characterized by inflamation and swelling of
the throat, as well as development of pus-filled
regions on the tonsils

Clinical Manifestations
Acute Streptococcus pyogenes infections

pharyngitis,
scarlet fever (rash)
impetigo
cellulitis, or erysipelas.
Invasive infections can result in
necrotizing fasciitis
myositis
streptococcal toxic shock syndrome
Patients may also develop immune-mediated sequelae such as acute
rheumatic fever and acute glomerulonephritis

Penicillin is usually administered to patients as soon as possible to quell the

possibility of the infection spreading from the upper respiratory system
into the lungs

Once in the lungs, the infection could give rise to pneumonia

Some cases also develop into rheumatic fever if left untreated (M types 1,
3, 5, 6, 18) associated with streptococcal pharyngitis; HS II )
Acute glomerulonephritis (M12 serotype) associated with streptococcal
pharyngitis or cutaneous infections ; HS III

Pathogenesis
Streptococci are members of the normal flora

Virulence factors of group A streptococci :

M protein and lipoteichoic acid for attachment
M protein (100 types) after antigen variability
subdivided in class I and II
M like proteins -antiphagocytic
F protein and lipoteichoic acid bind to host cells fibronectin

hyaluronic acid capsule (inhibits phagocytosis-strains responsible for severe systemic

infections )

other extracellular products, such as 4 pyrogenic (erythrogenic) heat labile toxin, which
causes the rash of scarlet fever SpeA, SpeB, SpeC, SpeF

streptokinase(lyses blood clots ) facilitates spread of bacteria in tissues

streptodornase (DNase B) (depolymerizes cell free DNA in purulent material )

Streptolysins (SLO-immunogenic,SLS nonimmunogenic: lyses leukocytes , plateletes ,

erythrocytes , stimulates release of lysosomal enzymes)

Some strains are nephritogenic. Immune-mediated sequelae do not reflect dissemination of

bacteria. Nongroup A strains have no defined virulence factors.
 Host Defenses

Antibody to M protein -type-specific immunity to group A streptococci

Antibody to erythrogenic toxin -prevents the rash of scarlet fever
Maternal IgG protects the neonate against group B streptococci.

Epidemiology
spread by respiratory secretions and fomites
the incidence of both respiratory and skin infections peaks in childhood
infection can be transmitted by asymptomatic carriers
acute rheumatic fever was previously common among the poor;
susceptibility may be partly genetic
Symptoms of Scarlet Fever an infection with group A streptococcus bacteria.
the bacteria make a toxin (poison) that can cause
the scarlet-colored rash from which this illness
gets its name.
The rash not all streptococci bacteria make this toxin and
not all kids are sensitive to it.
begins looking like a bad sunburn with tiny bumps and it
Two kids in the same family may both have strep
may itch infections, but one child (who is sensitive to the
usually appears first on the neck and face, often leaving toxin) may develop the rash of scarlet fever while
a clear unaffected area around the mouth the other may not.
it spreads to the chest and back, then to the rest of
the body
in body creases, especially around the underarms and
elbows, the rash forms classic red streaks.
areas of rash usually turn white when you press on them
By the sixth day of the infection the rash usually fades,
but the affected skin may begin to peel

fever above 101? Fahrenheit (38.3? Celsius)

swollen glands in the neck
the tonsils and back of the throat may be covered with a
whitish coating, or appear red, swollen, and dotted with
whitish or yellowish specks of pus
early in the infection, the tongue may have a whitish or
yellowish coating
chills, body aches, nausea, vomiting, and loss of appetite
 The scarlet fever rash usually fades on the sixth day after sore throat
symptoms began, but skin that was covered by rash may begin to peel
This peeling may last 10 days. With antibiotic treatment, the infection itself is
usually cured with a 10-day course of antibiotics, but it may take a few weeks
for tonsils and swollen glands to return to normal.
In rare cases, scarlet fever may develop from a streptococcal skin infection like
impetigo. In these cases, the child may not get a sore throat

Treating Scarlet Fever

a throat culture (a painless swab of throat
secretions) to see if the bacteria grow in the
laboratory
Once a strep infection is confirmed, the
doctor will likely prescribe an antibiotic for
child to be taken for about 10 days
Impetigo
a bacterial infection of the skin most commonly occurring between the
ages of 2 and
the infection tends to have a greater chance of occurrence in children
whose skin is compromised by previous irritants
These irritants can include poison ivy, insect bites or skin allergies
related to soap or makeup allergies.
Nonsuppurative Streptococcal Disease
Rheumatic Fever
Is characterized by inflammatory changes involving the heart , joints, blood
vessels, subcutaneous tissues
Involvment of the heart manifests as pancarditis ( endocarditis , pericarditis ,
myocarditis ), often associated with subcutaneous nodules
Joint manifestations can range from arthralgias to frank arthritis with multiple
joints involved
Specific M types (1,3,5,6,18) cause the disease
Is associated with streptococcal pharyngitis but not cutaneous infections
Is most common in young school-age children , occurs primarily during the fall
or winter

The diagnosis is made on the baseis of clinical findings and documented

evidence of a recent S. pyogenic infection such as :
1. Culture results
2. Detection of the group A antigen
3. Elevation of anti SLO (ASO); anti-DNase B; anti hyaluronidase antibodies

Acute Glomerulonephritis
Is characterized by acute inflammation of the renal glomeruli with edema ,
hypertension , hematuria , proteinuria

Diagnosis is determined on the basis of the clinical presentation and the finding
of evidence of a recent S. pyogenes infection
Progressive , irreversible loss of renal function has been observed in adults
 Alpha hemolysis: erythrocytes not lysed,
LABORATORY but hemoglobin altered to produce a green-
INDICATIONS: brown discoloration
Beta hemolysis: erythrocytes completely
lysed; the yellow base color of agar becomes
Gram stained smears are useless in S.
visible.
pharyngitis because viridans S. are members of the Gamma hemolysis: no hemolysis.
normal flora
from skin lesions , wounds are diagnostic
Serologic
cultures of swab on blood agar-show small ,
translucent , beta hemolytic colonies in 18-48 hours ASO titers are elevated in patients suspected of
having rheumatic fever
inhibited by bacitracin disk likely to be group A
The anti Dnase B test should be performed if
Streptococci streptococcal glomerulonephritis is suspected
PYR test :differentiation between beta hemolytic
streptococci : the presence of the enzyme L-
pyrrolidonyl arylamidase at S. pyogenes and absence
for S. anginosus
The catalase test distinguishes Staphylococci
from Streptococci and Enterococci. Positive catalase test. Negative catalase test.
(Performed on a (Performed on a colony of
colony of Streptococcus pyogenes.)
Staphylococci produce catalase, an enzyme that breaks Staphylococcus
down hydrogen peroxide into water and oxygen gas. aureus.)

Streptococci and enterococci do not produce catalase.

This is the first test you should do to identify an
unknown Gram-positive coccus.

If an isolate is catalase-positive, use the coagulase

test to separate S. aureus from other staphylococci.
If an isolate is catalase-negative, use hemolysis
pattern and biochemical tests to identify streptococci
and enterococci.

The test i s simple - pick up a colony with a sterile loop and emulsify it in a drop of hydrogen peroxide on a microscope slide. If the bacterium produces catalase, bubbles of oxygen will appear. (Avoid picking up fragments of agar - erythrocytes contain catalase.)
 Bacitracin sensitivity
distinguishes S. pyogenes
(= Group A)
from other beta-
hemolytic streptococci.

To peform the test - streak a plate, for

S. pyogenes on sheep blood agar
with bacitracin disc.
(Note absence of growth and beta-
hemolysis in a circular zone around
the disc)
single colonies, with a beta-hemolytic
isolate.
In the 'confluent zone' place a paper 'A
disc' impregnated with the antibiotic
bacitracin, using sterile forceps. Incubate
overnight at 37oC. [Disc is labeled 'A' for
'Group A strep'.]
Growth of S. pyogenes will be inhibited
in a zone around the disc.
Growth of other beta-hemolytic isolates
will not be inhibited.

S. agalactiae on sheep blood agar with bacitracin disc.

No zone of inhibition around disc.
GROUP B- S. Agalactiae
this bacterium has been the causative agent in mastitis in cows
currently, it has been found to be a cause of sexually transmitted urogenital infections in
females
proper diagnosis is necessary for women nearing labor because the infection can easily spread
to the child via the birth canal

S agalactiae may cause:

meningitis, neonatal sepsis, pneumonia in neonates
adults may experience vaginitis, puerperal fever, urinary tract infection, skin infection, and
endocarditis.

LABORATORY INDICATIONS:
CAMP + S. gr. B produce a diffusible , heat stable protein CAMP factor that enhances
beta hemolysis of S. aureus
Beta-hemolysis
The CAMP test identifies
Streptrococcus agalactiae (= Group
B).
Basis of the test: synergy between hemolysins of S.
agalactiae and S. aureus.
[Named with the initials of bacteriologists who devised it:
Christie, Atkins, Munch-Peterson.]
To peform the test - place a wide streak of S. aureus down
the center of a blood agar plate.
Make perpendicular streaks of an unknown isolate, with
known Group A and Group B isolates as controls. Where
hemolysins of S. agalactiae and S. aureus overlap, there will
be an 'arrowhead' or 'half-moon' of intense hemolysis.

CAMP Test: Vertical streak

of S. aureus; horizontal
streak of unknown isolate on
right; controls at left: Group
B S. agalactiae (upper) and
Group A S. pyogenes (lower).

Note 'arrowhead' of hemolysis

where hemolysins of S.
agalactiae
and S. aureus overlap;
unknown confirmed as S.
agalactiae.
GROUP D
many are harmless, the pathogenic strains cause complications of the human digestive tract
This group has recently been reclassified into two divisions: Enterococcus and non-Enterococcus.
The Enterococci include E. faecalis, a cause of urinary tract infections,
E. faecium, a bacterium resistant to many common antibiotics.
Diseases such as septicemia, endocarrditis, and appendicitis
Fecal matter from infected individuals is a source for isolation and identification techniques. Once
identified, Group D Strep can be treated with ampicillin alone or in combination with gentamicin.

LABORATORY INDICATIONS:
Hydrolysis of bile esculin (dark brown medium)
-this indicates the ability of the bacteria to tolerate bile from the liver
Growth in high salt conc.

Enterococcus faecalis ME
The Bile-Esculin Test identifies
Group D organisms.

Basis of the test: Ability to grow in the presence

of bile and hydrolysis of the glycoside esculin.

To peform the test - Touch a well-isolated colony

with a sterile loop and streak the surface of a Bile-
Esculin slant; incubate at 37oC overnight.
All group D organisms are members of the normal
intestinal flora and grow in the presence of bile,
which destroys many other bacteria.
Group D organisms hydrolyze esculin into
esculetin and glucose. In the presence of ferric
ions (present in the medium) esculetin forms a
black complex.

To distinguish Enterococci from Streptococcus

bovis, a salt-tolerance test is done.

To save time, inoculate both tests the same time.

Bile-esculin slants.
Left to right: Enterococcus;

Streptococcus bovis; Blank (no

bacteria); Staphylococcus
epidermidis, Staphylococcus
aureus
 NaCl Tolerance
distinguishes
Enterococci from S.
bovis.
Basis of the test: Ability to grow in
the presence of 6.5% NaCl.
To peform the test - Touch a well-
isolated colony with a sterile loop and
inoculate a tube of broth containing
6.5% NaCl; incubate overnight at 37oC.
Enterococci grow in this medium but
Streptococcus bovis does not.
Visible turbidity is evidence of growth.

NaCl-broth cultures.
Left to right: Enterococcus; Streptococcus bovis; Blank
(no bacteria); Staphylococcus epidermidis , Staphylococcus
aureus.
Enterococci produce visible turbidity but S. bovis does not.
Both staphylococci grow in high-salt medium thus this
test provides useful information only on a catalase-
negative isolate.
 Streptococcus pneumoniae = pneumococcus
referring to its morphology and its consistent involvement in pneumonia

Pneumonia is a disease of the lung that is caused by a variety of bacteria including :

Streptococcus
Staphylococcus
Pseudomonas
Haemophilus
Chlamydia
Mycoplasma,
several viruses
certain fungi and protozoans
The disease may be divided into two forms:

bronchial pneumonia :
is most prevalant in infants, young children and aged adults
S.pneumoniae; involves the alveoli contiguous to the larger
bronchioles of the bronchial tree

lobar pneumonia :is more prone to occur in younger adults; more

than 80% of the cases of lobar pneumonia are caused by
Streptococcus pneumoniae.
Lobar pneumonia involves all of a single lobe of the lungs (although
more than one lobe may be involved), wherein the entire area of
involvement tends to become a consolidated mass, in contrast to
the spongy texture of normal lung tissue.
OTHER IMPORTANT STREP
S. pneumoniae its surface carbohydrate antigens do not correspond to a specific Lancefield
group, Although not given a letter designation,
S. pneumoniae can be considered a Pyogenic (pus-producing) strain of Strep. It can be
distinguished from other Pyogenic bacteria by its :
high sensitivity to Optochin (no growth zone of inhibition). This bacterium causes pneumonia
meningitis, and otitis media. It also demonstrates alpha-hemolytic growth on blood agar.

IF direct

Viridans Group
The Viridans Streptococci, consisting of S. mutans and S. mitis, are alpha-hemolytic
bacteria. These bacteria inhabit the mouth. In fact, a large percentage of tooth decay can
be attributed to S. mutans
Cultivation
Streptococcus pneumoniae

is fastidious bacterium, growing best in 5% carbon dioxide

Nearly 20% of fresh clinical isolates require fully anaerobic conditions

In all cases, growth requires a source of catalase (e.g. blood) to neutralize the large amount
of hydrogen peroxide produced by the bacteria. In complex media containing blood, at 37C,
the bacterium has a doubling time of 20-30 minutes.

On agar, pneumococci grow as glistening colonies, about 1 mm in diameter.

Two serotypes, types 3 and 37, are mucoid.

. The transparent colony type is adapted to colonization of the nasopharynx, whereas the
opaque variant is suited for survival in blood. The chemical basis for the difference in colony
appearance is not known, but significant difference in surface protein expression between the
two types has been shown.

is a fermentative aerotolerant anaerobe

is usually cultured in media that contain blood

Special tests such as inulin fermentation, bile solubility, and optochin (an antibiotic)
sensitivity must be routinely employed to differentiate the pneumococcus from Streptococcus
viridans.
Streptococcus pneumoniaeGram-stain of blood broth culture
Streptococcus pneumoniae :contains within itself the enzymatic ability to disrupt and to disintegrate
the cells. The enzyme is called an autolysin.
The physiological role of this autolysin is to cause the culture to undergo a characteristic autolysis
that kills the entire culture when grown to stationary phase.
Autolysis is consistent with changes in colony morphology. Colonies initially appear with a plateau-type
morphology, then start to collapse in the centers when autolysis begins.

Sputum Gram stain from a patient with

Streptococcus pneumoniae Gram-stain a pneumococcal pneumonia
of blood broth culture

.
Identification

bile or optochin sensitivity

Gram-positive staining
hemolytic activity: cause alpha hemolysis on agar containing horse, human, rabbit and sheep
erythrocytes. Under anaerobic conditions they switch to beta hemolysis caused by an oxygen-
labile hemolysin.
Typically, pneumococci form a 16-mm zone of inhibition around a 5 mg optochin disc
undergo lysis by bile salts (e.g. deoxycholate). Addition of a few drops of 10% deoxycholate at
37C lyses the entire culture in minutes. The ability of deoxycholate to dissolve the cell wall,
depends upon the presence of an autolytic enzyme, LytA. Virtually all clinical isolates of
pneumococci harbor the autolysin and undergo deoxycholate lysis.

Streptococcus pneumoniae A mucoid strain on blood agar showing alpha hemolysis (green
zone surrounding colonies). Note the zone of inhibition around a filter paper disc
impregnated with optochin. Viridans streptococci are not inhibited by optochin.

Serotyping
The quellung reaction (swelling reaction) forms the basis of serotyping and relies on the
swelling of the capsule upon binding of homologous antibody
The test consists of mixing a loopful of colony with equal quantity of specific antiserum and
then examining microscopically at 1000X for capsular swelling.
Although generally highly specific, cross-reactivity has been observed between capsular types
2 and 5, 3 and 8, 7 and 18, 13 and 30, and with E. coli, Klebsiella, H. influenzaeType b, and
certain viridans streptococci.

 Treatment
Penicillin
Cephalosporins
Erythromycin , chloramphenicol , vancomycin are used for
patients allergic to Penicillin

Immunization
with 7 valent conjugated vaccine is recommended for all
children younger that 2 years of age
A 23- valent polysaccharide vaccine is recommended for
adults at risk for disease