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.
Most streptococci 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)
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

10.2. GRAM POSITIVE COCCI - GENUS STREPTOCOCCUS
Bacteria General
characteristics cocci G+,
=1-2m, arranged in chains
catalase -, facultative
anaerobe
Streptococcus pyogenes
(group A)1 microaerophilic
Clinical diseases
Suppurative infections
-pharyngitis
-scarlet fever
-cellulitis
-necrotizing fasciitis
Nonsuppurative infections
-Rheumatic fever ( M types
1, 3, 5, 6, 18) associated with
streptococcal pharyngitis; HS
II4
-Acute glomerulonephritis
(M12 serotype) associated
with streptococcal
pharyngitis or cutaneous
infections ; HS III 5
Reservoir/ Virulence factors Laboratory diagnosis/
transmission Treatment
Skin , M protein (100 types) after antigen variability - Specimens-throat swab, pus, blood
oropharynx subdivided in class I and II
in small M like proteins -antiphagocytic Smears- cocci G+, in chains
number / F protein and lipoteichoic acid bind to host cells
respiratory fibronectin Culture-
droplets, hyaluronic acid capsule (some strains) enriched blood agarS colonies = 1-2
direct contact systemic infections mm, hemolysis
Enzymes Identification-
Streptokinase A/B-lyses blood clots Rapid antigen test kit for agglutination
Deoxyribonucleases A-D- liquefies pus of the reactions
abscess and facilitates spread in tissues Bacitracin sensitive
C5a peptidase-degraded C5a PYR test +6 ( presence of pyrrolidonyl
Hyaluronidase-spreading factor arylamidase)
Hemolysins Serologic ASO test-titers of antibodies
against SLO > 200 is significant)
Streptolysin S -lyses leukocytes, platelets, Treatment
erythrocytes , nonimmunogenic Penicillin G, Penicillin V Erythromicin
Streptolysin O - lyses leukocytes, platelets, Prevention - Penicillin - in rheumatic
erythrocytes , immunogenic fever patients to prevent recurrence of the
Toxins disease
Erythrogenic exotoxin are responsible for the
rash in scarlet fever -is produced by lysogenic
strains/ Dick test3
Pyrogenic(Spes A/B/C/F)
exotoxin A toxic shock syndrome
exotoxin B- necrotizing fasciitis
Cell surface structure of S pyogenes and extracellular substances
 GROUP A
the first group in the Lancefield classification system includes only
one species of Streptococcus, 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 may take the form of :
pharyngitis,
scarlet fever (rash)
impetigo
cellulitis, or erysipelas.

Invasive infections can result in necrotizing fasciitis, myositis and

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
Other diseases linked to S. pyogenes are skin infections such as
impetigo, cellulitis, and erysipelas.
Pathogenesis
Streptococci are members of the normal flora.
Virulence factors of group A streptococci include :
M protein and lipoteichoic acid for attachment
a hyaluronic acid capsule that 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 ), and
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 gives type-specific immunity to group A streptococci. Antibody to
erythrogenic toxin prevents the rash of scarlet fever. Immune mechanisms are important in
the pathogenesis of acute rheumatic fever. Maternal IgG protects the neonate against group
B streptococci.

Epidemiology
Group A -hemolytic streptococci are 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. Group B streptococci are common in the normal
vaginal flora and occasionally cause invasive neonatal infection.
Symptoms of Scarlet Fever
The rash
is the most striking sign of scarlet fever begins looking like a
bad sunburn with tiny bumps and it may itch
usually appears first on the neck and face, often leaving a clear Scarlet fever is caused by an infection with group A
unaffected area around the mouth streptococcus bacteria. The bacteria make a toxin (poison)
It spreads to the chest and back, then to the rest of the body. that can cause the scarlet-colored rash from which this
In body creases, especially around the underarms and elbows, the illness gets its name.
rash forms classic red streaks. Areas of rash usually turn white Not all streptococci bacteria make this toxin and not all
when you press on them kids are sensitive to it. Two kids in the same family may
By the sixth day of the infection the rash usually fades, but the both have strep infections, but one child (who is sensitive
affected skin may begin to peel to the toxin) may develop the rash of scarlet fever while
the other may not.
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

When scarlet fever occurs because of a throat infection, the

fever typically stops within 3 to 5 days, and the sore throat
passes soon afterward. 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
When scarlet fever occurs because of a throat infection, the
fever typically stops within 3 to 5 days, and the sore throat
passes soon afterward. 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
Impetigo is a bacterial infection of the skin most commonly
occurring between the ages of 2 and 6. The bacterial
infection that causes Impetigo is most often related to the
streptococcus or staphylococcus aureus bacterias. 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, eczema, insect bites
or skin allergies related to soap or makeup allergies.
Impetigo
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
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

LABORATORY
INDICATIONS:
Gram stained smears are useless in S. pharyngitis
because viridans S. are members of the normal flora
from skin lesions , wounds are diagnostic
cultures of swab on blood agar-show small ,
translucent , beta hemolytic colonies in 18-48 hours
inhibited by bacitracin disk likely to be group A
Streptococci
PYR test :differentiation between beta hemolytic
streptococci : the presence of the enzyme L-
pyrrolidonyl arylamidase at S. pyogenes and absence
for S. anginosus
Catalase -
Alpha hemolysis: erythrocytes not lysed, but
hemoglobin altered to produce a green-
brown discoloration
Beta hemolysis: erythrocytes completely
lysed; the yellow base color of agar becomes
visible.
Gamma hemolysis: no hemolysis.
Serologic
ASO titers are elevated in patients suspected of
having rheumatic fever
The anti Dnase B test should be performed if
streptococcal glomerulonephritis is suspected
The catalase test distinguishes Staphylococci
from Streptococci and Enterococci.
Positive catalase test. Negative catalase test.
(Performed on a (Performed on a colony of
Staphylococci produce catalase, an enzyme that breaks colony of Streptococcus pyogenes.)
down hydrogen peroxide into water and oxygen gas. Staphylococcus
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 is 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

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
S. pyogenes on sheep blood agar
'Group A strep'.]
with bacitracin disc.
Growth of S. pyogenes will be inhibited
(Note absence of growth and beta-
in a zone around the disc.
hemolysis in a circular zone around
Growth of other beta-hemolytic isolates
the disc)
will not be inhibited.

S. agalactiae on sheep blood agar with bacitracin disc.

No zone of inhibition around disc.
GROUP B
The B classification of Lansefield also includes only one bacterium, S. Agalactiae
For years 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. Although infection is easily treated with penicillin, 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, and 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
Type D Streptococcus is the next clinically important bacterium because of the multitude of diseases it is
known to cause. Although 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, and E. faecium, a
bacterium resistant to many common antibiotics. Diseases such as septicemia, endocarrditis, and
appendicitis have also been attributed to group D Strep.
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
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
(obviously!), 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.
Hemolysins
pneumococci secrete exotoxins
Two hemolysins have been described, the most potent of which is pneumolysin.
Pneumolysin is stored intracellularly and is released upon lysis of pneumococci by autolysin.
Pneumolysin binds to cholesterol and thus can indiscriminately bind to all cells without
restriction to a receptor
This protein assembles into oligomers to form transmembrane pores which ultimately lead to
cell lysis.
Pneumolysin can also stimulate the production of inflammatory cytokines, inhibit beating of
the epithelial cell cilia, inhibit lymphocyte proliferation, decrease the bactericidal activity of
neutrophils, and activate complement
A second hemolysin activity has been described but has not been identified. In addition,
pneumococci also produce hydrogen peroxide in amounts greater than human leukocytes
produce. This small molecule is also a potent hemolysin.
 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