STREPTOCOCCI

Dr John Egbagba
FmcPath
 Streptococci General criteria
• Gram positive, cocci, arrange in pairs or chains

• Nutritionally fastidious, require complex media supplemented with blood or serum

• Most are facultative anaerobe, but do not use O2 metabolically

• Some are capnophilic and others are anaerobic

• Ferment carbohydrate and produce lactic acid.

• No gas production

• Catalase negative, Non motile

 Staphylococcus Streptococcus

• Arrange in cluster • Arrange in chain

• Grow in ordinary media • Require enriched media

• Colony – pin head, large • Colony - small, pin point

• Selective media – Mannitol salt agar • Selective media - Crystal Violet Blood Agar
• Catalase – positive • Catalase – negative
• Produce thick pus • Produce thin pus
• Penicillin – usually resistant
• Penicillin – usually sensitive
Staphylococcus Streptococcus
Streptococcus pneumoniae Streptococcus pyogens
 Classification of Streptococci
1.Hemolysis

-hemolysis: incomplete lysis of RBC with the formation

of green pigment.

-hemolysis: complete hemolysis

No hemolysis

2.Lancefield classification: a serologic classification (A to

V)

3.Biochemical reactions: are used for species that can not

be classified into the Lancefield classification
(nongroupable), e.g. viridans streptococci.
 Hemolysis
Trait Alpha hemolysis Beta Hemolysis Gamma
Hemolysis
Type incomplete Complete No
Color Greenish discoloration Clear zone No change
Margin Indefinite Sharp define
Area of zone 1-2 mm wide 2-4 mm wide
Mechanism H2O2 produce by bacteria oxidizes HB Hemolysin Completely lyse
and produce biliverdin (green) RBC

example Viridans streptococci (viridis = green) S. Pyogen Enteroccocci

S. Pneumonae S. agalactiae
 Lancefield Grouping
Based on C carbohydrate Ag on bacterial cell wall. 21 Lancefield group A-W
Lancefield Groups
• Membrane Ag
• Groups A-H, K-V
• Group A
• Strep. pyogenes
• Group B
• Strep. agalactiae
• Group C, F, G
• Strep equisimilis
• Strep zooepidemicus
• Strep anguinosus
• Group D
• E. faecalis
• Strep bovis
• Strep equines
• Group K
• Strep salivarius
• Viridans
• Strep mutans
• Strep mitis
• Strep sanguis
• Pneumococci
 S. Pyogens
• Gram positive, spherical / oval cocci

• Non-motile, non sporing

• 0.5 to 1 µm in diameter

• They divide in a plane perpendicular to the long axis of the chain

• Form long chain, when recovered from liquid culture

• May arrange in short chain, clusters or pairs in gram stains of samples from
infected tissue
 Antigenic structure
A. Capsule

• Many strains produce hyaluronic acid capsule

• such strains grow as mucoid colony on Blood Agar,
• These are highly virulent
• Anti-phagocytic effect
• Non-immunogenic (chemically Identical to hyaluronic acid of host so, not
recognize as foreign)
B. Cell wall composition –

• Outer layer of protein and lipoteichoic acid

• Middle layer of group specific C carbohydrate (rhamnose & N
acetylglucosamine)

• Inner layer of peptidoglycan

M protein

• Most important virulence factor

• determines the type of S. pyogenes
• Appears as hair-like projections on Streptococcal cell wall
M Protein

• Antiphagocytic activity

• S. pyogenes is not infectious in the absence of M Protein

• It is antigenic and antibody to M Protein provides type specific immunity

• More than 100 different types of M Proteins have been identified by

serological means

• Individual may suffer from recurrent infections with strains expressing

different versions of M Protein
M protein
Depending on M protein there are

• Rheumatogenic strain – Causes primarily rheumatic fever

• Nephritogenic strain- Causes primarily AGN

 Enzymes
A. Streptokinase (Fibrinolysin)

• Two types – Streptokinase A & B

• Catalyses conversion of plasminogen to plasmin it then cleaves fibrin and fibrinogen

• Result in lyses of clots and fibrin deposits, thrombi and emboli

• Facilitate rapid spread of S. pyogenes in infected tissues by breaking the fibrin barrier

• In contrast, S. aureus aims at hiding behind a wall of fibrin and produce a localized abscess

• It is used for treatment of pulmonary emboli and of coronary artery and venous thrombi

• Anti-streptokinase antibody are useful marker of infection

B. Deoxyribonucleases / Streptodornase / DNAase

• Four types (Dnase) are produced – DNAase A, B, C, D

• DNase B is the most common form
• They liquify DNA (thick) which was released from dead neutrophil and
make the pus thin

• Thus act as ‘spreading factor’ by liquefying purulent exudates

• Antibiotics thus gain better access and infection recover quickly
B. Deoxyribonucleases / Streptodornase / DNAa
Use

• Mixture of streptokinase and streptodornase are used to liquefy exudate and

facilitate removal of pus & necrotic tissue

• Anti Dnase B is of great value in diagnosing AGN following skin infection,

not sore throat (streptolysin O is irreversibly inhibited by skin cholesterol;
so S. pyogenes fail to produce ASO in cutaneous infections.)
C. Hyaluronidase

• Spreading factor.

• Streptococcus causes spreading type of skin infection because this enzymes

breaks hyaluronic acid, the ground substance of connective tissue.

• Strains produce large amount of hyaluronidase (M4, M22) are non-

capsulated.
 Toxins
A. Hemolysin / Streptolysin

Two types- Streptolysin O and Streptolysin S

Streptolysin O

• Oxygen labile, heat labile, Immunogenic.

• Hemolytic in reduce state.

• Responsible for complete hemolysis around the colony. Inhibited by

cholesterol
Mechanism

• Lyse RBC = Binds with cholesterol containing cell membrane leads to

production of holes

• Degranulation and lysis of PMNs

• Inhibit phagocytosis by macrophage

• Cytotoxic for platelet and cardiac tissues

Antisterptolysin O

• Antibody titre of Streptolysin O, (ASO) >160-200 units use for diagnosis of

recent streptococcal infection (pharyngeal)

• ASO block hemolysis by streptolysin O

• Skin infection does not induce ASO.

Why ASO response following streptococcal skin infection is low

Inactivation of SLO by cholesterol present in skin.

Streptolysin S:

• Oxygen stable.

• Cell-bound, not antigenic (low molecular wt˂20,000 D).

• Produced in the presence of serum. S indicate serum stable

• Responsible for hemolysis in media.

Mechanism

• Interact with membrane phospholipid in exerting toxic effect

• Can lyse erythrocytes, leukocytes and platelets.

• Kills phagocytes by releasing the lysosomal contents after engulfment.

Pyrogenic (Erythrogenic) Exotoxins

• Produced by both the scarlet fever strains and new invasive S. pyogenes strains.

• Four serologically distinct toxins (Spe A, B, C and F).

Spe A and C called erythrogenic toxin.

• Responsible for scarlet fever; extensive rash.

• Antibody prevents rash.

• Associated with streptococcal toxic shock syndrome, necrotizing fasciitis

(exotoxin B) rash of scarlet fever.
Pyrogenic (Erythrogenic) Exotoxins

• They are superantigens (except for Spe B, which is a cysteine protease) and may
exhibit the following biological activities:
• Enhances release of pro-inflammatory cytokines, e.g. IL-1,2, TNF-α , INF-γ
(pyrogenicity)

• causes skin rash

• Inflammation
• Hypotensive shock
• Organ failure
• Immuno-suppression
 Diseases by S. pyogens

Fig- rash of scarlet fever Fig- Necrotizing fascitis

 Necrotizing fasciitis: (“flesh-eating bacteria”)
Infection deep in subcutaneous tissues that spreads along fascial planes,
destroying muscle and fat

• Initially cellulitis followed by bullae (fluid filled blisters; bulla is singular),

gangrene, systemic toxicity, multi-organ failure.

• Prompt medical intervention is essential.

 Scarlet fever
• If streptococci produce erytrogenic toxin

• Frequently develop scarletina rash on upper chest

spreading to extremities within 1 to 2 days after the
initial clinical symptoms of pharyngitis develops.

• The rash disappears over the next 5 to 7 days,

followed by desquamation.

• strawberry tongue a characteristic lesion

 Streptococcal Toxic Shock Syndrome (TSS)
• Occurs in invasive and bactaraemic infection, particularly in necrotizing
fasciitis.

• Multisystem toxicity following soft tissue infection progressing to shock and

organ failure.

• This disease results from release of pyrogenic exotoxin A and B.

• Superantigen, stimulate T cell and release cytokines that mediate shock and
tissue injury.
 Acute rheumatic fever (ARF)
• Develops within 2-3 weeks following pharyngitis.

• Streptococcal skin infection do not cause RF.

• Autoimmune disease

Pathogenesis: cross-reactivity of heart and joint tissues with antibody formed

against M protein (Type II Hypersensitivity).

Prevention- if treated with penicillin within first 10 days following initiation of

pharyngitis.
 Acute rheumatic fever (ARF)

• Commonly involved M type 1, 3, 5, 6, 18.

• Inflammatory reaction characterized by fever, arthritis, carditis, chorea,

erythema marginatum or subcutaneous nodules.

• ASO titre and ESR elevated

• Morbidity & mortality linked to subsequent disease of heart valve (Rheumatic

Heart Disease)

• Recurrences common, lifelong penicillin therapy needed.

 Acute Glomerulonephritis

• Acute inflammation of renal glomeruli

• Occur after either pharyngeal or skin infection (most common)

• Following 2-3 weeks of respiratory (pharyngitis) or 1-2 weeks of cutaneous

(pyoderma) streptococcal infection.

• Associated with M types 2, 4, 12 & 49 (most frequent) .

 Acute Glomerulonephritis

• Granular accumulations of immunoglobulin due to deposition of immune

complexes on the basement membrane of kidney (Type III Hypersensitivity)

• Rheumatic fever can be reactivated by recurrent streptococcal infections,

whereas nephritis does not
 Lab diagnosis

A. Specimen:

• Can be obtained from throat or skin

• Swab, pus, sputum, blood.
• Serum obtained for antibody detection.

B. Gram staining and microscopy

• Gram positive cocci arranged in chains or pairs with pus cells.

C. Culture (Definitive diagnosis)
• small pinpoint, dome shaped colonies surrounded by a large zone of beta
haemolysis in blood agar.
• Encapsulated cells produce mucoid colonies
D. Biochemical test Catalase negative. Differentiates from Staphylococcus
Bacitracin test:

• To distinguishing between Group A beta-hemolytic streptococci (bacitracin POS)

and other beta-hemolytic streptococci that are isolated from pharyngeal swabs

• 95% sensitivity for Grp A strep

• When grown on blood agar, Group A streptococci are sensitive to (killed by) the
antibiotic bacitracin

• Bacitracin Sensitive - S.pyogenes

• Bacitracin Resistant - S.agalactiae

E. rapid test Antibody detection

• Detect S. pyogenes directly in throat swab without culture.

• Diagnostic kits use specific antibody to detect group specific Abs to detect group A CHO Ag
in the swab material.

Anti DnaseB

• Positive following streptococcal skin infection. Important in diagnosing AGN.

ASO titre

• Titre high soon after Group A streptococcal infection-within 3 to 4 wks

• Indicate previous infection, as the time of development of RF culture is usually negative.

• Unreliable in AGN.
Treatment

• Oral penicillin V or amoxicillin for streptococcal pharyngitis.

• For penicillin-allergic patients, oral cephalosporin or macrolides can be used.

• For severe systemic infection- combined use of I/V penicillin and aminoglycosides
recommended.

• Patient with a history of rheumatic fever require long term penicillin therapy as
prophylaxis to prevent recurrence of the disease.
 STREPTOCOCCUS AGALACTIAEEPTOCOCCUS
• Str.AGALACTIAE
agalactiae belongs to Lancefield group B.
• Its primary human habitat is the colon.
• It may be carried in the throat and, importantly, 10–40% of
women intermittently carry Str. agalactiae in the vagina.
• Previously, Str. agalactiae was recognized primarily as a
cause of bovine mastitis (agalactia, want of milk).
• However, since 1960 it has become the leading cause of
neonatal infections in industrialized countries and is also an
important cause of morbidity among peripartum women and
non-pregnant adults with chronic medical conditions.
• Among -haemolytic streptococci, Str. agalactiae is the most
frequent isolate from blood cultures.
Pathogenesis/Virulence factors
• Str. agalactiae produces several virulence factors, including
haemolysins, capsule polysaccharide, C5a peptidase
• (only human pathogenic strains), hyaluronidase (not all
• strains), and various surface proteins that bind human IgA and serve
as adhesins.
• Nine different types of the capsular polysaccharide have been
identified (Ia, Ib, and II-VIII).
• The serotype most frequently associated with neonatal infections is
type III, whereas infections in adults are more evenly distributed over
the different serotypes.
• Among the haemolysins produced by Str. agalactiae, one, known as
the CAMP factor (so-called because it was originally described by
Christie, Atkins and Munch-Petersen), plays an important role in the
recognition of this species in the laboratory.
• The CAMP factor lyses sheep or bovine red blood cells pre-
treated with the -toxin of Staph. aureus
• Purified CAMP factor protein is lethal to rabbits when
injected intravenously
 CAMP Factor Test
S. aureus
Group B (Spingomyelinase C)
Streptococcus
(CAMP Factor)

Group A
Streptococcus

Enhanced
Zone of
Hemolysis
CLINICAL DISEASES OF S. agalactiae
S. agalactiae may colonize the lower gastrointestinal tract and
genitourinary tract
1. Neonatal sepsis or meningitis
Early-onset (during the first week of life): infection acquired in utero or at
birth. Pneumonia is common in addition to meningitis.
Late-onset (older infants): infection acquired from an exogenous source.
(Premature infants are at greater risk.)
2. Infection of pregnant women
Urinary tract infections, amnionitis, endometritis, and wound infections
3. Infection in men and nonpregnant women
Patients are generally older and have underlying conditions.
Bacteremia, pneumonia, bone and joint infections, skin and soft tissue
infections. Mortality is higher.
Viridans streptococci
(-hemolytic or nonhemolytic, most are nongroupable; they, except
for S. suis, are divided into 5 subgroups based on the specific
diseases they cause)
These streptococci colonize the oropharynx, GI tract, and GU tract;
rarely on the skin surface.
Diseases:
Subacute endocarditis (group: Mitis)
Intra-abdominal infections (group: Anginosus)
Dental caries (group: Mutans)
Cariogenicity of S. mutans is related to its ability to synthesize
glucan from fermentable carbohydrates (e.g. sucrose) as well as
to modify glucan in promoting increased adhesiveness.
 Enterococci
(E. faecalis, E. faecium)
Physiological properties are similar to the streptococci.

Form large colonies on blood plate;

most are nonhemolytic.

Microscopic morphology is similar to

S. pneumoniae.

Resistant to 6.5% NaCl, 0.1% methyl

blue and grow in bile-esculin agar.
More resistant to antibiotics than the
streptococci.

Colonize the large intestine of

humans and animals. An opportunist.
 Pneumococci

• Formerly called Diplococcus pneumonia.

• This was one of the first pathogenic bacteria observed by Gram stain.
 Morphology
• Gram-positive cocci, non-motile, encapsulated (polysaccharide)

• Lancet shaped (oval with somewhat pointed ends)

• Arranged in pair or short chains in specimen.

• It has fastidious nutritional requirements and can grow only on enriched media
supplemented with blood products.

• Produce alpha hemolysis on blood agar

• Ferment carbohydrates, producing lactic acid as the primary metabolic byproduct.

 Structure

Capsular polysaccharide (SSS)

• Virulent strains are covered with a complex polysaccharide capsule.

• currently, 94 serotypes are recognized.
• Encapsulated strains are pathogenic whereas
• Noncapsulated strains are avirulent.
• It has antiphagocytic and antigenic activity.
• Provide type specific immunity.
 Capsular swelling (Quellung) reaction
Pneumococcal capsule reacts with the type specific antiserum and becomes
apparently swollen and become more easily visible under the microscope.
 Structure

Cell wall carbohydrate

• The peptidoglycan layer is typical of gram-positive cocci.

• ‘C’ polysaccharide or C substance exposed on cell surface - It

precipitates a serum globulin fraction [CRP].
 Toxin

Pneumolysin

• It is a cytosolin similar to streptolysin O.

• It retain within the cytosol of intact pneumococci.
• It is released by autolysin from interior of the bacterium.
• Binds with cholesterol in mammalian host cell membrane and creates pore.
• It may contribute to pathogenesis by destroying ciliated epithelial cells and
phagocytic cells.
 Toxin
Autolysin

• Hydrolase/ amidase present in inactivated form in cell wall.

• Activated by surface active substances, β-lactam antibiotics, bile or
aging and results in cell lysis.

• Autolysis of aged cells produce colonies with depressed center

(Draughtsman colony).
 Enzymes
IgA proteases

• It enhances the ability of the organism to colonize the mucosa of the

upper respiratory tract by inactivating secretory IgA Ab

Hyaluronidase

• Facilitate spread of the organism

 Diseases
• Pneumonia

• Bronchopneumonia – secondary infection following viral infection

• Meningitis – pneumococcus spread from the pharynx to meninges via blood

because of coexistent bacteremia

• Bacteremia and sepsis

• Sinusitis and otitis media

• Other suppurative infection- empyema, pericarditis, suppurative arthritis.

 Causes of pneumonia
• S. pneumoniae

• S. aureus

• K. pneumonia

• S. pyogens

• H. influenzae

• Fusobacterium species

• L. pneumophilia

• Bacteroids melaninogenicus

• Anaerobic cocci (Peptostreptococcus, peptococcus)

 Lab diagnosis
Specimen

• According to site of lesion

• Blood, Sputum, nasopharyngeal swab, CSF, Pus, aspirate from sinus

Smear preparation and microscopy

• Gram staining - Demonstration of gram positive cocci, lancet shaped

diplococci surrounded by an unstained halo of capsule
 Lab diagnosis
Culture

• Streptococcus grows in enriched

media like Blood agar media with
5-10% CO2 at 37 C

• Young culture of encapsulated

bacteria produce circular,
glistening, dome-shaped colonies.
 Lab diagnosis
Biochemical reaction

• Bile solubility test- undergo rapid autolysis when exposed to bile on an

isolated colony

• Optochin sensitivity- sensitive. A zone of inhibition is seen around the

colony

• Ferments glucose, lactose, sucrose and inulin giving acid only.

 Treatment
• Penicillin is the drug of choice for susceptible strain

• For resistant strains and penicillin-allergic patients, cephalosporin,

erythromycin, chloramphenicol or vancomycin can be used.

• Serious pneumococcal diseases should be treated with combination of

antibiotics. Vancomycin with ceftriaxone commonly used as empiric
treatment.
 immunization
Pneumococcal conjugate vaccine (PCV13)

• Recommended for all children younger than 5 years old, all adults 65
years or older, and people 6 years or older with certain risk factors.

Pneumococcal polysaccharide vaccine (PPSV23)

• Recommended for all adults 65 years or older. People 2 to 64 years of age

who are at high risk of pneumococcal disease should also receive PPSV23.
QUESTIONS?