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Classification of Streptococcus

I. Basis of Classification

A. Haemolysis on Blood Agar (Brown's Classification)

B. Lancefield Serological Grouping (by Cell Wall Carbohydrate Antigen)

C. Physiological/Biochemical Classification

• Viridans streptococci - heterogeneous group; 5 subgroups: Anginosus, Mitis (includes S. pneumoniae), Mutans, Salivarius, Bovis
• Enterococci - formerly Group D, now a separate genus (Enterococcus)
• S. pneumoniae - α-haemolytic, bile-soluble, optochin-sensitive

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Streptococcus pyogenes (Group A Streptococcus)

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II. Virulence Factors

A. Surface/Structural Components

1. Hyaluronic Acid Capsule

• Outer layer; antigenically identical to human connective tissue hyaluronic acid
• Prevents phagocytic recognition (non-immunogenic - treated as "self" by host)
• Mucoid strains with prominent capsules cause more severe systemic infections (e.g., toxic shock syndrome)

2. M Protein (Most Important Virulence Factor)

• The dominant type-specific antigen; >200 serotypes (emm types) based on the variable N-terminal amino terminus
• Consists of two alpha-helical polypeptide chains anchored in the cytoplasmic membrane, protruding above the cell surface
• Functions:
  • Resists phagocytosis by inhibiting C3b deposition and complement activation
  • Mediates adherence to pharyngeal epithelial cells (via fibronectin)
  • Mediates adherence to skin keratinocytes
  • Binds IgG (Fc region), blocking opsonisation
• Class I M proteins share exposed shared antigens - only these strains cause rheumatic fever
• Class II M proteins do not share exposed antigens
• Anti-M antibodies are type-specific and protective; immunity to one type confers no protection against others

3. M-Like Proteins (Protein H, IgG-binding proteins)

• Encoded by the emm gene superfamily (>20 genes)
• Bind the Fc region of IgG, blocking opsonisation

4. Lipoteichoic Acid (LTA)

• Embedded in the cell wall; complexes with M protein to mediate binding to fibronectin on epithelial cells
• Involved in adherence to nasopharyngeal mucosal cells

5. Protein F (Fibronectin-binding protein)

• Mediates adhesion to epithelial cells and Langerhans cells in skin
• Expression is regulated by environmental O₂/CO₂ levels

6. Peptidoglycan

• Structural cell wall component
• Activates complement (alternative pathway) and triggers inflammation

B. Secreted Enzymes (Spreading Factors)

1. Streptolysin O (SLO)

• Oxygen-labile, cholesterol-sensitive cytolysin
• Lyses RBCs, WBCs, platelets
• Immunogenic - anti-SLO antibodies (ASO titre) form and are diagnostically important
• ASO titre >200 IU/mL in adults suggests recent streptococcal infection

2. Streptolysin S (SLS)

• Oxygen-stable, non-immunogenic haemolysin
• Responsible for β-haemolysis seen on blood agar plates
• Lystes RBCs, WBCs, platelets; can damage cardiac muscle

3. Streptokinase (Fibrinolysin)

• Dissolves fibrin clots; activates plasminogen → plasmin
• Facilitates spread through tissue barriers
• Two types: streptokinase A and B
• Immunogenic; anti-streptokinase antibodies are produced

4. Hyaluronidase ("Spreading factor")

• Hydrolyses hyaluronic acid in ground substance of connective tissue
• Facilitates spread of organisms through tissues
• Immunogenic; anti-hyaluronidase antibodies formed

5. DNases (Deoxyribonucleases A, B, C, D)

• Depolymerise DNA, reducing viscosity of pus (which contains DNA from lysed leukocytes)
• Anti-DNase B titre is especially useful in diagnosing skin infections (impetigo)

6. NADase (Nicotinamide Adenine Dinucleotidase)

• Destroys NAD; cytotoxic effect on leukocytes
• Possibly interferes with intracellular killing

7. Neuraminidase

• Cleaves neuraminic acid from glycoproteins on cell surfaces
• Aids colonisation and spread

8. C5a Peptidase

• Cleaves and inactivates the complement fragment C5a
• Impairs neutrophil chemotaxis, reducing local defence

C. Toxins

1. Erythrogenic (Pyrogenic) Exotoxins (Sperotoxins A, B, C)

• Also called streptococcal pyrogenic exotoxins (Spe A, B, C)
• Bacteriophage-encoded (SpeA and SpeC are phage-encoded; SpeB is chromosomally encoded)
• Act as superantigens: bypass normal antigen processing, directly stimulate large populations of T cells (up to 20% of all T cells) by binding MHC class II molecules and T-cell receptor Vβ region simultaneously → massive cytokine release (IL-1, IL-2, TNF-α)
• SpeA and SpeC are primarily responsible for Streptococcal Toxic Shock Syndrome (STSS)
• SpeB (cysteine protease): degrades host tissues, cleaves IgG, inactivates C5a
• Erythrogenic toxin causes the diffuse red rash of Scarlet fever (by direct skin vessel damage and hypersensitivity)

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III. Infections Produced by S. pyogenes

A. Suppurative (Pyogenic) Infections

1. Pharyngitis ("Strep Throat") - Most Common

• Infection of the posterior pharynx and tonsils
• Abrupt onset: high fever, severe sore throat, dysphagia, exudates, cervical lymphadenopathy
• Children aged 5-15 most affected; spread by respiratory droplets
• Diagnosis by throat swab culture; rapid antigen test

2. Scarlet Fever

• Pharyngitis + erythematous rash caused by erythrogenic toxin (SpeA)
• Rash: diffuse, fine, erythematous ("sandpaper" texture), begins on trunk, spares the face (circumoral pallor), face shows flushing with Pastia's lines (rash in skin creases), followed by desquamation
• Strawberry tongue - initially white-coated with red papillae, then becomes red
• Caused by strains that produce pyrogenic exotoxin; immunity is toxin-type specific, not anti-M

3. Impetigo (Pyoderma)

• Superficial skin infection, more common in children in warm/humid climates
• Begins as vesicles that become pustules and then honey-coloured crusted lesions
• Can be complicated by post-streptococcal glomerulonephritis (not rheumatic fever)

4. Erysipelas

• Acute infection of the superficial dermis and cutaneous lymphatics
• Sharply demarcated, raised, bright red, warm, tender advancing border
• Most commonly affects the face and lower limbs; caused by lymphatic spread

5. Cellulitis

• Deeper infection involving dermis and subcutaneous fat
• Less defined margins compared to erysipelas
• Features: erythema, warmth, oedema, tenderness

6. Necrotising Fasciitis ("Flesh-eating" disease)

• Deep infection along fascial planes; extensive destruction of muscle and fat
• Introduced via minor cuts, burns, surgery, or varicella vesicles
• Progresses rapidly: cellulitis → bullae → gangrene → systemic toxicity → multiorgan failure
• Must be managed with emergency surgical debridement + antibiotics
• High mortality even with treatment; bacteraemia common

7. Streptococcal Toxic Shock Syndrome (STSS)

• Multisystem toxicity caused by M serotypes 1 and 3 with prominent mucoid capsules
• SpeA and SpeC superantigens drive massive cytokine release
• Features: soft-tissue pain, fever, chills, nausea/vomiting → shock → multiorgan failure (renal, pulmonary, hepatic, cardiac)
• Differs from staphylococcal TSS: most patients are bacteraemic and many have necrotising fasciitis

8. Other Suppurative Infections

• Puerperal (childbed) fever: uterine infection post-delivery or post-abortion; can progress to septicaemia
• Pneumonia: uncommon; typically post-influenza
• Otitis media: rarely primary
• Lymphangitis: red streaks along lymphatics from the primary infection site
• Septicaemia: particularly from wound or skin infections

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B. Non-Suppurative (Immunological) Sequelae

1. Acute Rheumatic Fever (ARF)

• Follows pharyngitis only (not skin infection)
• Latent period: 2-4 weeks after pharyngitis
• Pathogenesis: Antibodies against M protein cross-react with cardiac myosin and valvular tissue (molecular mimicry)
• Diagnosis by Jones Criteria (2 major + evidence of prior streptococcal infection, or 1 major + 2 minor)
• Major criteria: Carditis, Polyarthritis, Chorea (Sydenham's), Erythema marginatum, Subcutaneous nodules
• Repeated episodes cause rheumatic heart disease (mitral stenosis most common)
• Only strains with Class I M proteins cause ARF

2. Post-Streptococcal Glomerulonephritis (PSGN)

• Follows either pharyngitis (latent period 10 days) or impetigo (latent period 3 weeks)
• Only certain nephritogenic strains (limited M types)
• Immune complex deposition in glomeruli (type III hypersensitivity)
• Features: oedema (peri-orbital first), hypertension, haematuria (smoky urine), proteinuria, reduced serum complement (C3)
• Course is usually benign and self-limiting in children; rarely progresses to renal failure

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IV. Laboratory Diagnosis of S. pyogenes

A. Specimen Collection

B. Direct Microscopy (Gram Stain)

• Gram-positive cocci in chains
• Useful in pus from sterile sites; not helpful from throat (due to commensal streptococci)
• No capsule visible on Gram stain (hyaluronic acid capsule is not detected)

C. Culture

1. Media

• Sheep Blood Agar (SBA) - preferred medium; incubated anaerobically or with 5% CO₂ (anaerobic conditions enhance β-haemolysis)
• Todd-Hewitt Broth - enrichment broth for fastidious organisms
• Incubation: 37°C for 24-48 hours

2. Colony Characteristics

• Small (1-2 mm), translucent/grey-white colonies
• Large, clear zone of β-haemolysis surrounding the colony (complete lysis of RBCs)
• Growth inhibited in high glucose media

3. Growth in Broth

• Long chains; produces turbidity

D. Identification Tests

1. Catalase Test

• S. pyogenes: Catalase-negative (distinguishes from Staphylococci which are catalase-positive)

2. Bacitracin Sensitivity (Presumptive Test)

• S. pyogenes is sensitive to bacitracin (0.04 U disk) - zone of inhibition present
• Other β-haemolytic streptococci (Groups B, C, G) are resistant
• Sensitivity: ~95% for Group A; simple and inexpensive screening test

3. PYR Test (L-Pyrrolidonyl β-Naphthylamide Hydrolysis)

• S. pyogenes is PYR-positive (produces pyrrolidonyl arylamidase)
• Enterococci are also PYR-positive; other streptococci are negative
• More specific than bacitracin for definitive Group A identification

4. Lancefield Grouping (Definitive Test)

• Cell wall carbohydrate extracted by acid hydrolysis or enzyme treatment
• Identified by precipitation, latex agglutination, or coagglutination with Group A-specific antibodies
• Gold standard for definitive identification

5. CAMP Test

• S. pyogenes: Negative (distinguishes from Group B which is CAMP-positive)

6. Hippurate Hydrolysis

• S. pyogenes: Negative

E. Rapid Antigen Detection Tests (RADT)

• Detect Group A carbohydrate antigen directly from throat swab in 5-10 minutes
• Specificity ~99%, Sensitivity ~70-90%
• A positive RADT is diagnostic; a negative result in clinical suspicion should be followed by culture
• Based on latex agglutination or EIA

F. Serological Tests (Used for Diagnosing Sequelae)

1. Anti-Streptolysin O (ASO) Titre

• Measures antibodies against Streptolysin O
• Elevated in pharyngitis and ARF (titre >200 Todd units/mL in adults; >333 in children)
• Not elevated in skin infections (skin lipids neutralise SLO, preventing immune response)
• Rises 1-3 weeks after infection; peaks at 3-5 weeks; returns to normal in 6 months
• A single high titre or a rising titre indicates recent group A streptococcal infection

2. Anti-DNase B (ADB) Titre

• Measures antibodies against Streptococcal DNase B
• Elevated in BOTH pharyngitis AND skin infections (impetigo)
• Most sensitive test for post-impetigo PSGN
• Used when ASO is negative (especially in skin infections)

3. Anti-Hyaluronidase (AHT) Titre

• Elevated after skin infections
• Used as an adjunct

4. Streptozyme Test

• Slide agglutination test detecting antibodies to multiple streptococcal antigens (SLO, DNase B, streptokinase, hyaluronidase)
• Used as a screening test; less specific than individual titres

G. Molecular (PCR) Diagnosis

• Multiplex PCR detects S. pyogenes directly from clinical specimens
• Used in throat swabs, blood, sterile sites
• Higher sensitivity than culture, especially in partially treated patients
• Available in reference laboratories; not routine in all settings

H. Summary Table of Identification

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V. Treatment (Brief)

• Drug of choice: Penicillin G / Amoxicillin (10 days for pharyngitis to prevent ARF)
• Alternative: Erythromycin or Azithromycin (penicillin allergy)
• S. pyogenes remains uniformly sensitive to penicillin (no resistance reported)
• Necrotising fasciitis: Penicillin + Clindamycin (Clindamycin inhibits toxin production) + surgical debridement
• Long-term penicillin prophylaxis to prevent ARF recurrence

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