Streptococcaceae
Streptococcaceae family
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Classification - Includes 7 genera:
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Streptococcus – a major cause of human infections
Enterococcus – frequent cause of human infections
Aerococcus – occasionally isolated from
endocarditis, meningitis, or urinary tract infection
Lactococcus – rare human pathogen
Pediococcus – NF of lower GI tract, occasional
isolate from abscesses
Leuconostoc – infrequently isolated from blood,
wounds, or abscesses
Gemella – rare isolate of upper RT infection
Streptococcaceae family
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We will focus only on Streptococci and Enterococci
Preliminary differentiation is based on the hemolytic
pattern they exhibit when grown on blood agar (,,
or )
Serologic classification – was developed in the
1930’s by Lancefield.
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based on a precipitin reaction between a group specific
antigen (C substance) which is a carbohydrate extracted
from the cell wall, and specific antisera.
test divides the Streptococci into serologic groups A
through U.
Some Strep don’t possess a group specific antigen and
can’t be identified by this method.
Streptococcaceae family
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Lancefield typing is used today primarily to
distinguish between beta hemolytic Strep.
Physiologic or biochemical characteristics can be
used to differentiate species within the Streptococci
and Enterococci genera and will be discussed later.
In the future DNA probes and hybridization or PCR
related tests will be used to differentiate the
different species of Streptococci and Enterococci .
Streptococcaceae family
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Morphology and General Characteristics
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Gram positive cocci that appear in pairs or chains
(most often seen when grown in broth culture)
because cell division occurs in only 1 plane.
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The length of the chain depends upon the firmness of
attachment of the individual cells.
Strep pneumoniae is a lancet shaped diplococcus.
Strep quickly lose the ability to retain the crystal violet in
the Gram stain as they age and will appear G- in old
cultures.
Gram stain of Strep. species grown
in broth
Strep pneumomiae
Streptococcaceae family
Many form capsules
 Streptococci are more fastidious than
Staphylococci and require an infusion media
or an enriched media such as blood agar to
grow.
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Many are hemolytic on blood agar and the
source of the blood (sheep vs. rabbits, etc.) may
affect the type of hemolysis that the organism
exhibits.
 Clinical labs all use 5% sheep RBCs in their
blood agar.
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Types of hemolysis
Streptococcaceae family
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The type of atmosphere in which the organism is
grown may affect the type of hemolysis of Group A,
beta hemolytic Strep. They produce two types of
hemolysins:
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Streptolysin O which is oxygen labile
Streptolysin S which is oxygen stable.
Most strains produce both streptolysin S and O, but a few
only produce streptolysin O which would not be detected if
the organism is grown in the presence of oxygen.
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Stabbing the primary isolation plate after inoculation may
provide the lower oxygen tension necessary to identify those
strains that only produce streptolysin O.
Streptococcaceae family
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Addition of trimethoprim-sulfamethoxazole (1.25 ug
and 23.75 ug, respectively) to sheep blood agar
may be used to suppress the growth of NF and
allow better recovery of beta hemolytic Strep from
cultures with a lot of NF such as those from the
oropharynx.
Strep are microaerophilic and for good growth
require reduced oxygen tension such as what is
found in a candle jar or in a CO2 incubator.
Nutritionally deficient Strep may require vitamin B6
or pyridoxal for growth and may be found satelliting
Staph aureus.
Streptococcaceae family
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Strep pneumoniae is sensitive to the cold and may
fail to grow in cultures that have been refrigerated.
Biochemical identification
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Differentiation from other genera
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Other G+ cocci - Strep are catalase -, while Staph and
Micrococci are catalase +
Other cocci – Neisseria are G- cocci, but since Strep
quickly lose their G + characteristic as they age, they may
be confused with Neisseria.
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However, Neisseria are oxidase + and Strep are oxidase –.
Streptococcaceae family
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Differentiation within the genus
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Once a clinical isolate is identified as a Streptococcus sp.,
before biochemical identification is done several things
should be considered:
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Source of the specimen
 hemolytic Strep isolated from the respiratory tract are
likely to be viridans group Strep which are NF, or Strep
pneumoniae which may be a pathogen
 hemolytic Strep isolated from the GI tract is probably
the NF Enterococcus.
 hemolytic Strep isolated from the respiratory tract are
likely to be Strep pyogenes (group A)
 hemolytic Strep isolated from the genitourinary tract
are likely to be Strep agalactiae (group B).
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Streptococcaceae family
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Differential diagnosis – certain Strep are more
commonly associated with specific clinical syndromes
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group A with sore throat
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group B with neonatal meningitis
Hemolysis – if the organism is beta hemolytic, test for
Lancefield types A, B, C, D, F, and G
Streptococcaceae family
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Group A (Strep pyogenes) biochemical
differentiation (is  hemolytic)
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Susceptibility to .04 units of bacitracin (done only on
beta hemolytic Strep). Group A is sensitive and
occasionally groups B, C or G are sensitive.
SXT disks
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Resistance to SXT – both Groups A and B are
resistant, others are sensitive
Pyr test
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Production of pyroglutamyl aminopeptidase (pyr) –
both group A Strep and Enterococci are +, others,
including group B, are -.
Streptococcaceae family
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Group B (Strep agalactiae) biochemical
differentiation (is  hemolytic)
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CAMP (Christie, Atkins, Munch-Peterson) test – tests
for a diffusible, heat stable extracellular protein that
enhances the hemolysis of sheep RBCs by the beta
hemolysin of S. aureus.
 The synergistic action of the two hemolysins gives
an arrowhead of hemolysis.
 This test is + for group B and – for all others.
CAMP test
Streptococcaceae family
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Hydrolysis of hippurate – due to the enzyme hippuricase.
Group B is + while occasionally Enterococci and viridans
group Strep are +, all others are -.
Growth in 6.5% NaCl – Most group B are + as is
Enterococcus, all others are -.
Resistance to SXT – both Groups A and B are R, others are
S
Group D biochemical differentiation (may be , , or 
hemolytic). Includes both Enterococci and nonEnterococci species
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Bile esculin hydrolysis – all group D grow in the presence of
bile esculin and are + for esculin hydrolysis, all other groups
are -
Bile esculin hydrolysis
positive
negative
Streptococcaceae family
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To differentiate group D Enterococci from non-Enterococci:
Pyr - + for Enterococci only
Growth in 6.5% NaCl - + for Enterococci only
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To differentiate viridans group Streptococci from Strep
pneumoniae (both are  hemolytic):
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Optochin (ethyl hydrocupreine hydrochloride) susceptibility –
optochin is a detergent that activates an autolytic enzyme
that lyses the cell wall of Strep pneumoniae.
Only Strep pneumoniae is S, viridans group Strep are R.
Optochin disk (taxo P) test
Streptococcaceae family
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Bile solubility- bile salts also activate the autolytic
enzyme of Strep pneumoniae.
 Strep pneumoniae is soluble, viridans group Strep
are not.
The viridans group Strep don’t contain group specific
C antigens and can’t be identified by serological tests.
Most labs don’t speciate the viridans group Strep
except for isolates from blood cultures or for
epidemiologic purposes.
Others ways to identify Streptococci
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Determination of serogroups – Lancefield
typing based on C substance in cell wall
Streptococcaceae family
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Determination of serotypes – Group A strep are
divided into >80 different serotypes based on the
type of M protein that they express.
Quellung test – used in identifying the >84 different
serotypes of Strep pneumoniae – is based on
differences in the type of specific soluble substance
(SSS) in their capsule.
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In the quellung reaction when specific antisera against
SSS binds to the SSS, under the microscope it appears as
if the capsule has swelled.
This is due to a change in the refractive index that occurs
when an antibody binds to its specific antigen.
Quellung reaction
Streptococcaceae family
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Mechanisms of pathogenicity
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Strep pyogenes – extracellular products
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Streptolysin S – alters membrane permeability and lyses
RBCs, leukocytes, and other cells containing sterols in
their membranes.
Streptolysin O – forms membrane penetrating channels
(porin) leading to membrane defects and cell lysis.
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In addition to RBCs, it is also cytotoxic to many tissue cells,
and is particularly cardiotoxic.
It is antigenic and during strep throat infections, but not
during skin infections, antibodies are formed.
An anti-streptolysin O titer (ASOT) can be used to
diagnose a recent streptococcal infection.
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Streptococcaceae family
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Erythrogenic toxins – Act as superantigens and cause
fever, are cardiotoxic, cause enhanced susceptibility to
endotoxin shock, suppress antibody responses, and are
indirectly responsible for the rash seen in scarlet fever.
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The rash is due to a delayed hypersensitivity reaction to
other streptococcal antigens that is enhanced by the toxin.
Toxin may be involved in causing a toxic shock like
syndrome similar to the toxic shock syndrome caused by
Staph. aureus.
Streptokinase – causes lysis of fibrin clots by cleaving
plasminogen to plasmin which then acts as a protease to
lyse fibrin clots.
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Used in medicine to prevent heart attacks
Streptococcaceae family
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Hyaluronidase
DNAse – degrades accumulated inflammatory exudate
DNA from leukocyte disintegration.
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Is antigenic and antibodies against it can be used to
diagnose recent Strep infections, including skin infections.
Strep pyogenes – cell associated factors
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M protein is a fibrous protein on the cell surface. Its
presence is essential for virulence (it is the MAJOR
virulence factor for group A Strep) and there are over 80
different types.
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is poorly immunogenic
Antibodies, if made, are protective, long lasting, and type
specific.
Streptococcaceae family
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M protein protects the Strep from opsonization and,
therefore, phagocytosis, by binding to fibrinogen.
may also play a role in adherence.
Some types of M protein can act as superantigens causing
shock in the affected patient.
Structure of Group A cell wall
Streptococcaceae family
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Lipoteichoic acid (LTA) – mediates attachment to epithelial
cells
Hyaluronic acid capsule (note that this organism also
produces a hyaluronidase).
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Antibodies to the capsule are not protective against
infection.
Strep agalactiae – extracellular products
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Hemolysin
Toxin that either directly or indirectly causes an increase in
vascular permeability in the lungs leading to respiratory
distress
Neuraminidase – cleaves sialic acid residues and may be
involved in hyalin membrane disease (a lung problem)
Hyaluronidase
Streptococcaceae family
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Strep agalactiae – cell associated factors
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DNAse
Protease
Capsule – antibodies are type specific and protective
LTA – for attachment
Other beta hemolytic Strep –
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Some group C produce streptolysin O or S, streptokinase,
or a capsule
Some group G produce streptolysin O or S, streptokinase,
DNAse, erythrogenic toxins or M protein
Streptococcaceae family
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Strep pneumoniae – extracellular products
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Pneumolysin O – is cytolytic for RBCs and other tissue
cells.
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is actually a cytoplasmic rather an extracellular protein.
is only released when the bacteria lyse.
acts as a porin to disrupt host cell membranes.
Leukocidan
Neuraminidase
Hyaluronidase
IgA protease - cleaves IgA
Strep pneumoniae – cell surface structures
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Capsule – is the most important virulence factor.
there are > 80 different types and antibodies against the
capsule are type specific and protective.
Strep pneumoniae with mucoid colonies due to
capsule
Streptococcaceae family
Clinical significance – Strep are responsible for a wide
variety of diseases.
 Many of the infections are characterized by pus
producing lesions (suppurative) and manifestations
suggesting toxemia (toxin involvement).
 Infants tend to be more susceptible to Strep infections
with prolonged, low grade infections.
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Rarely do nephritis and rheumatic fever follow.
In contrast, older children and adults tend to get more
acute and self-limited infections, with nephritis and
rheumatic fever as occasional complications (these
are non-suppurative sequelae to be discussed later).
Streptococcaceae family
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Beta hemolytic groups A, B, and C are the
most virulent Strep, with the greatest number
of Strep infections due to group A.
 Most alpha hemolytic and non hemolytic Strep
are much less virulent.
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Alpha hemolytic Strep (viridans group Strep) are
found as NF in the RT and GI tract.
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They are the most common cause of subacute bacterial
endocarditis.
In contrast, Strep pneumoniae infections are a
leading cause of death throughout the world,
particularly in the elderly and the very young.
Streptococcaceae family
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Strep pyogenes infections
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Streptococcal pyoderma or impetigo – is a superficial skin
infection characterized by a vesicular rash that becomes
pustular and in later stages is covered with a thick crust.
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Staph aureus may be a secondary invader.
is endemic in tropical areas and is caused by M serotypes
different than those that cause pharyngitis.
is spread by close, direct contact
disease starts with skin colonization, followed by invasion
through abrasions
type specific antibodies to M are made, but antibodies to
streptolysin O are uncommon
The most serious complication is acute glomerulonephritis
which may occur 2-3 weeks post infection.
Impetigo
Strep pyogenes infections
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Erysipelas – a diffuse, erythematous skin
infection that is most often on the face and
occurs following pharyngitis
Strep pyogenes infections
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Wound infections – occasionally causes wound infections
following trauma. Usually due to simple contamination of
the wound.
Cellulitis – an infection of subcutaneous tissue which may
be accompanied by lymphangitis and abscess formation.
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Patients typically exhibit chills, fever, and marked signs of
toxicity.
Necrotizing fasciitis (”flesh eating bacteria”) – when there
is destruction of the fibrous tissue deep in the skin.
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The sheath of tissue that covers the muscle is destroyed.
Destruction is due to release of exotoxin A that stimulates
production of cytokines damaging endothelial lining and
leaking fluid into the extravascular space causing diminished
blood flow, tissue hypoxemia, and tissue death.
Can also be caused by Staph aureus and other bacteria.
Necrotizing fasciitis
Necrotizing fasciitis
Flesh removed to stop spread
Gangrene
Strep pyogenes infections
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Myositis – is a result of Strep invasion of muscle
tissue resulting in extensive muscle necrosis and
overwhelming sepsis. This condition is usually
fatal.
Strep pyogenes infections
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Pharyngitis – Strep or sore throat – most common disease
caused by Strep pyogenes.
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Is characterized by an abrupt onset of sore throat, malaise,
headache and fever.
The throat (pharynx) is erythematous (red) with a grayishwhite exudate.
The tonsils and lymph nodes are enlarged and the tonsils
may become infected chronically.
Spread to the sinuses and middle ear may also occur.
Disease is spread by droplets of respiratory secretions or via
food or water.
Secondary effects may include rheumatic fever (to be
discussed later).
Disease is usually self-limited, but penicillin should be used
to prevent secondary complications from occurring.
Pharyngitis
Strep pyogenes infections
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Scarlet fever – results from URT infection with group A
Strep that has been lysogenized by a phage which carries
the gene for the erythrogenic toxin.
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Symptoms are similar to pharyngitis plus an erythematous
rash that first appears on the upper chest and spreads to the
rest of the body.
Lasts about 1 week and is followed by several weeks of
extensive desquamation of the skin.
The tongue is also affected with red spots on a yellowishwhite colored tongue (strawberry) followed by peeling and a
red beefy colored (raspberry) tongue.
Immunity to the toxin is demonstrated by the Dick test –
inject toxin and if local antibodies are absent, the patient will
develop a local erythema.
The erythrogenic toxin may also be involved in causing a
toxic-shock like syndrome.
Scarlet fever
Strep pyogenes infections
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Non-suppurative complications of Strep infections – no
purulent inflammatory response is seen and the diseases
can’t be diagnosed by culturing the organism which is no
longer present
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Rheumatic fever – is an inflammatory disease that may involve the
heart, joints, subcutaneous tissue, and the CNS.
It occurs primarily in children 6-15 years of age at a time period
of 1-5 weeks after Strep throat, but not pyoderma.
The major manifestations are polyarthritis, carditis, chorea, and
erythema marginatum.
Carditis can involve all layers of the heart (characteristic
lesions are called Aschoff bodies or nodules) and can lead to
rheumatic heart disease which is characterized by chronic,
progressive damage to the heart and possibly death.
The mechanism by which rheumatic fever is initiated is an
autoimmune phenomena involving cross-reacting antibodies
made against Strep, that react with self tissues.
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Rheumatic fever
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Antibody binding attracts host reactive cells,
complement gets activated and this leads to
cellular damage.
Diagnosis is done using ASOT assays.
This complication can be prevented by treating
Strep throat with penicillin.
Rheumatic fever
Strep pyogenes infections
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Acute glomerulonephritis – this may occur 2-3 weeks
following pharyngitis or pyoderma.
 It is characterized by edema, hypertension,
headache, malaise, circulatory congestion, and
hematuria.
 It is only associated with specific nephritogenic M
serotypes of group A Strep.
 It is probably an immune complex disease
resulting from deposition of circulating immune
complexes of Strep antigens and antibodies in the
basement membrane of the glomeruli of the
kidney.
 Damage is probably due to the attraction of host
reactive cells and subsequent complement
activation.
Streptococcaceae family
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Strep agalactiae (group B) infections – Group B
Strep are part of the NF of the adult vagina, urethra,
and rectum, and, occasionally, the pharynx.
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appears to be sexually transmitted.
In adults it rarely causes URT infections, meningitis,
bacteremia, and endocarditis.
It used to be a major cause of puerperal sepsis (postpartum sepsis).
The most serious cases of group B infections occur in the
newborn. There are two types of disease in the newborn.
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Early onset disease – occurs when babies become infected
in utero or at birth, with the organism gaining access through
the respiratory tract.
The disease has a high mortality rate and is
characterized by respiratory disease and bacteremia.
Septicemia and meningitis may also occur.
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Strep agalactiae (group B)
infections
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Late onset disease – occurs 7-10 days after birth and
the baby probably acquires the organism by direct
contact with mucosal surfaces.
 The disease is characterized by meningitis or
osteomyelitis and possibly bacteremia.
 The mortality rate is high, but lower than that for
the early-onset disease.
Other Streptococcal group infections –
Group C – occasionally causes diseases similar
to those caused by group A, but without the nonsuppurative sequelae complications.
 Group F – tend to form abscesses in patients
with underlying disease or trauma.
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Streptococcaceae family
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Group D Enterococci and non-enterococci – are part of the
NF of the GI tract, but are a common cause of UT
infections, subacute bacterial endocarditis, abdominal
abscesses, and wound infections.
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Finding Strep bovis in the blood suggests that the surfaces
of the colon have been compromised and this has been
associated with carcinoma of the colon.
An early screen for colon cancer?
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Group G – rarely associated with disease except in those
with underlying conditions such as tumors.
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In these individuals group G Strep can cause the same
diseases associated with groups A and B Strep
Streptococcaceae family
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Viridans group Strep – are NF of the RT, GI tract, and
genital tract.
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Are the most common cause of subacute bacterial
endocarditis where they colonize damaged heart valves.
Strep mutans plays a role in dental decay.
Strep pneumoniae – are found as part of the NF in
the URT in 15% of children and 5% of adults.
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This carrier state is sporadic.
It is probably a pathogen when it is the most predominant
organism isolated.
Pneumonia – is the leading cause of bacterial pneumonia,
particularly in the very young, the elderly, and those with
immunological deficiencies.
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Bacteremia is common and meningitis may occur.
The mortality rate is 5-7%.
Streptococcaceae family
Streptococcaceae family
Strep pneumoniae infections
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Meningitis – Not as common as other causes of
meningitis except in children under 1 year of age
where it is the second most common cause of
meningitis.
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The mortality rate in this group is high.
Otitis media – is the most common cause of
middle ear infection in children (1/3 of cases).
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May also cause purulent sinusitis and peritonitis.
Otitis media
Streptococcaceae family
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Antimicrobial susceptibility –
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Use penicillin for groups A, B, C, and G.
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Enterococcus and other group D – must do
sensitivity testing.
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Use erythromycin for individuals allergic to penicillin.
Susceptibility testing is not necessary.
There are now strains of Enterococcus faecalis that are
resistant to all known antibiotics.
Viridans group Strep – do sensitivity testing
Streptococcaceae family
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Strep pneumoniae – used to always use penicillin,
but now there are resistant strains, so sensitivity
testing is necessary.
Other treatments
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There is a polyvalent vaccine for strains of Strep
pneumoniae that are most likely to cause
pneumonia.
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is given to those most likely to be infected, i.e., young and
old individuals and those with chronic respiratory
problems.