Streptococcal Classification and Identification Tests

General Characteristics of Streptococci

This chapter is highlighted as a "MOST IMPORTANT CHAPTER", indicating the critical nature of the information for understanding streptococcal characteristics and identification.

Hemolysis Patterns

Hemolysis refers to the lysis (breakdown) of red blood cells by bacterial toxins. It is a key characteristic used in the preliminary identification of Streptococcus species, observed on blood agar plates.

• Beta-hemolysis ($\beta$-hemolysis): This indicates complete lysis of red blood cells, resulting in a clear zone around the bacterial colony. Two types of beta-hemolysis zones were observed:
  • Small zone of hemolysis: Approximately $6$ mm (e.g., associated with Group B Strep as per one image).
  • Bigger zone of hemolysis: Approximately $14$ mm (seen in comparison to the smaller zone, but specific organism not explicitly labeled for this size in the visible text).
• Alpha-hemolysis ($\alpha$-hemolysis): This indicates partial lysis of red blood cells, causing a green discoloration around the colony due to the oxidation of hemoglobin. Several Streptococcus groups exhibit alpha-hemolysis.
• Gamma-hemolysis ($\gamma-hemolysis): This signifies no hemolysis or no change in the red blood cells around the colony.

Classification of Streptococcus from Humans (Key Species and Groups)

The following table summarizes important Streptococcus species and groups, their Lancefield classifications, hemolytic patterns, and relevant comments for identification and clinical significance:

• S. pyogenes:
  • Lancefield Group: A
  • Hemolytic Pattern: $\beta$ (Beta-hemolytic)
• S. agalactiae:
  • Lancefield Group: B (Known as Group B Strep)
  • Hemolytic Pattern: $\beta$, $\gamma$ (Beta or Gamma-hemolytic)
• S. dysgalactiae subsp. equisimilis:
  • Lancefield Group: C, G
  • Hemolytic Pattern: $\beta$ (Beta-hemolytic)
  • Comments: Formerly S. equisimilis; pyogenic; associated with respiratory infections and Soft Tissue and Skin Infections (SSTI).
• S. pneumoniae:
  • Lancefield Group: None
  • Hemolytic Pattern: $\alpha$ (Alpha-hemolytic)
• S. bovis species group:
  • Lancefield Group: D
  • Hemolytic Pattern: $\alpha$, $\gamma$ (Alpha or Gamma-hemolytic)
  • Comments: Part of the Viridans streptococci; commonly associated with colon cancer and Infective Endocarditis (IE).
• S. mutans group:
  • Lancefield Group: Not useful for classification
  • Hemolytic Pattern: $\alpha$, $\gamma$, rarely $\beta$ (Alpha, Gamma, rarely Beta-hemolytic)
  • Comments: Part of the Viridans streptococci; a primary cause of dental caries and also associated with IE.
• S. salivarius group:
  • Lancefield Group: Not useful for classification
  • Hemolytic Pattern: $\alpha$, $\gamma$ (Alpha or Gamma-hemolytic)
  • Comments: Part of the Viridans streptococci; an opportunistic pathogen.
• S. mitis group:
  • Lancefield Group: Not useful for classification
  • Hemolytic Pattern: $\alpha$ (Alpha-hemolytic)
  • Comments: Part of the Viridans streptococci; associated with IE and opportunistic infections.
• S. anginosus group:
  • Lancefield Group: A, C, F, G, or no detectable group
  • Hemolytic Pattern: $\alpha$, $\beta$, $\gamma$ (Alpha, Beta, Gamma-hemolytic)
  • Comments: Part of the Viridans streptococci; formerly known as S. milleri; comprises three species: S. anginosus, S. constellatus, and S. intermedius; known for causing purulent infections (abscesses).

Key Biochemical Identification Tests

CAMP Test

• Purpose: Primarily used to identify Streptococcus agalactiae (Group B Strep).
• Principle: S. agalactiae produces a CAMP factor that acts synergistically with the $\beta$-lysin of Staphylococcus aureus to cause enhanced hemolysis on blood agar.
• Positive Result: An arrowhead-shaped zone of enhanced hemolysis forms at the intersection of the S. agalactiae streak and a perpendicular streak of S. aureus.

Bile Esculin Test

• Purpose: To differentiate bacteria that can hydrolyze esculin in the presence of bile.
• Principle: Organisms that produce esculinase can hydrolyze esculin to esculetin and glucose. Esculetin reacts with ferric ions in the medium to produce a black precipitate.
• Positive Result: Blackening of the medium, indicating esculin hydrolysis. (Generally positive for Enterococcus species and Group D streptococci).

Catalase Test

• Purpose: To detect the presence of the enzyme catalase, which breaks down hydrogen peroxide ($\text{H}<em>{2} ext{O}</em>{2}$) into water ($\text{H}<em>{2} ext{O}$) and oxygen ($\text{O}</em>{2}$).
• Principle: Most Streptococcus species are catalase-negative, while Staphylococcus species are catalase-positive.
• Positive Result: Production of visible bubbles (oxygen gas) immediately after adding hydrogen peroxide to a bacterial colony.

Indole Test

• Purpose: To determine if an organism can produce indole from the amino acid tryptophan.
• Principle: Bacteria possessing the enzyme tryptophanase hydrolyze tryptophan to indole, pyruvic acid, and ammonia. Indole is detected by Kovac's reagent.
• Positive Result: A red color develops in the reagent layer after adding Kovac's reagent.
• Negative Result: No color change or a yellow/brown color in the reagent layer.

Methyl Red (MR) and Voges-Proskauer (VP) Tests (MRVP)

• Purpose: Part of the IMViC battery of tests used to differentiate members of the family Enterobacteriaceae based on their glucose fermentation pathways.
• Methyl Red (MR):
  • Principle: Detects organisms that perform mixed acid fermentation, producing stable acidic end-products (e.g., lactic, acetic, formic acids) from glucose, resulting in a low pH.
  • Positive Result: The indicator (methyl red) turns red at a pH of $4.4$ or below.
  • Negative Result: The indicator remains yellow/orange at a pH above $6.2$.
• Voges-Proskauer (VP):
  • Principle: Detects organisms that utilize the butanediol pathway for glucose fermentation, producing neutral end-products such as acetoin and 2,3-butanediol.
  • Positive Result: A red color develops after adding reagents (e.g., Barritt's reagents, consisting of $\text{alpha}$-naphthol and potassium hydroxide) due to the oxidation of acetoin to diacetyl.
  • Negative Result: No color change or a copper color.

Nitrate Reduction Test

• Purpose: To determine if an organism can reduce nitrate ($\text{NO}<em>{3}$) to nitrite ($\text{NO}</em>{2}$) or further to gaseous nitrogen ($\text{N}_{2}$).
• Principle: Bacteria with the enzyme nitrate reductase convert nitrate to nitrite. Some further reduce nitrite to nitrogen gas. Detection involves adding reagents or zinc powder.
• Positive Result (no gas): Red color after adding reagents A (sulfanilic acid) and B (N,N-dimethyl-$\alpha$-naphthylamine), which indicates nitrite production. Alternatively, if no color develops after adding zinc powder, it means nitrate was completely reduced beyond nitrite.
• Positive Result (gas): Presence of a gas bubble in the Durham tube, indicating the reduction of nitrate to nitrogen gas.
• Negative Result: If, after adding reagents A and B, no color develops, and then adding zinc powder results in a red color. This means nitrate was still present and was reduced by the zinc, not by the bacteria.

Oxidase Test

• Purpose: To detect the presence of the enzyme cytochrome c oxidase, a component of the electron transport chain.
• Principle: The enzyme oxidizes a chromogenic reducing agent (e.g., tetramethyl-p-phenylenediamine) to form a colored product.
• Positive Result: A color change to purple/blue within $10-30$ seconds.

Hippurate Hydrolysis Test

• Purpose: A specific test to identify Streptococcus agalactiae (Group B Strep).
• Principle: Organisms possessing the enzyme hippuricase hydrolyze sodium hippurate into benzoic acid and glycine. Glycine can then be detected by specific reagents.
• Positive Result: A deep purple color, specifically indicative of Streptococcus agalactiae.
• Negative Result: The solution remains slightly yellow, pink, or colorless. (Enterococcus is given as an example of a negative result).

Triple Sugar Iron (TSI) Agar

• Purpose: A differential medium used to identify Gram-negative enteric bacilli based on their ability to ferment glucose, lactose, and/or sucrose, and to produce hydrogen sulfide gas.
• Principle: The medium contains three sugars (glucose, lactose, sucrose), pH indicators, and an iron salt. Different fermentation patterns lead to acid production (yellow color changes) in the slant and/or butt. Hydrogen sulfide production results in a black precipitate.
• Possible Results: A combination of changes over a $24$-hour incubation period, including yellowing of the slant/butt, gas production (cracks in the agar), and blackening (H$_2$S production), provide a characteristic biochemical profile for identification.