GRAM POSITIVE COCCI

Objectives

Discuss biochemical tests for identifying Gram-positive cocci.
Describe growth requirements and atmospheric conditions.
Differentiate colonial morphology on media.
Set up and interpret biochemical tests for identification.

Common Genera of Gram-Positive Cocci

Common: Enterococcus, Staphylococcus, Streptococcus.
Less Common: Abiotrophia, Aerococcus, Leuconostoc, Micrococcus, etc.
Viridans Streptococci: Includes S. mutans, S. salivarius, S. anginosus, S. mitis, S. bovis.

Gram Staining Characteristics

Staphylococcus spp.: Gram-positive cocci in clusters.
Micrococcus spp.: Gram-positive cocci in pairs and clusters.
Streptococcus/Enterococcus spp.: Gram-positive cocci in chains.

Cultivation Media Requirements

$5\%$ Sheep Blood Agar: Supports most non-fastidious organisms, detects hemolysis.
Chocolate Agar: Enriched for fastidious organisms.
CNA Agar: Selective for Gram-positive; contains colistin and nalidixic acid.
PEA Agar: Isolates Gram-positive from mixed flora.
Mannitol Salt Agar: Selective for staphylococci; distinguishes based on mannitol fermentation.

Hemolysis Types in Blood Agar

Alpha ( $\alpha$ ): Partial hemolysis, green discoloration.
Beta ( $\beta$ ): Complete lysis, clear zone.
Gamma ( $\gamma$ ): No hemolysis.

Lancefield Group Typing

Identifies streptococcal species based on group-specific antigens.

Additional Tests

CAMP Test: Enhances lysis of RBCs to identify Group B strep.
Optochin Test: Differentiates S. pneumoniae from other $\alpha$ -hemolytic streptococci.
PYR Test: Identifies Enterococcus and Group A strep by hydrolysis reaction.
Hippurate Hydrolysis and LAP: Enzyme tests for further identification.

Growth Requirements

$6.5\%$ NaCl Tolerance: Identifies Enterococcus spp.
Temperature Tolerance: Growth at $42^{\circ}C$ helps in Enterococcus identification.

Core Biochemical Identification Techniques

Catalase Test
- Biochemical Mechanism: The enzyme catalase neutralizes toxic hydrogen peroxide ( $2H<em>{2}O</em>{2}$ ) by breaking it down into water ( $H<em>{2}O$ ) and diatomic oxygen ( $O</em>{2}$ ). This protects the cell from oxidative damage.
- Results:
- Positive: Rapid effervescence (gas bubbles/ $O_{2}$ release).
- Negative: No bubbles.
- Organisms:
- Positive: Staphylococcus spp., Micrococcus spp.
- Negative: Streptococcus spp., Enterococcus spp.
Coagulase Test
- Biochemical Mechanism: Differentiates the highly pathogenic S. aureus from other staphylococci. It involves two forms of the enzyme:
- Bound Coagulase (Clumping Factor): Located on the cell wall; it acts directly on fibrinogen in plasma to convert it to insoluble fibrin, causing organisms to clump.
- Free Coagulase (Extracellular): Secreted enzyme that reacts with coagulase-reacting factor (CRF) to form a thrombin-like complex, which then converts fibrinogen to a fibrin clot.
- Results:
- Positive: Visible clumping (slide test) or clot formation in plasma (tube test).
- Negative: No clumping or clot remains liquid.
- Organisms:
- Positive: Staphylococcus aureus.
- Negative: Staphylococcus epidermidis, Staphylococcus saprophyticus (CoNS).
Mannitol Salt Agar (MSA)
- Biochemical Mechanism: This is a selective and differential medium. The high salt concentration ( $7.5\% NaCl$ ) creates high osmotic pressure that inhibits most non-halotolerant bacteria. The differential aspect relies on mannitol fermentation; bacteria that can metabolize mannitol produce organic acid byproducts, lowering the pH of the medium.
- Results:
- Positive: Medium changes from red/pink to yellow (pH shift detected by phenol red).
- Negative: Medium remains red/pink (no fermentation).
- Organisms:
- Positive: Staphylococcus aureus (typically), some S. saprophyticus.
- Negative: Staphylococcus epidermidis.
Microdase Test (Modified Oxidase)
- Biochemical Mechanism: Detects the presence of cytochrome c in the respiratory chain. The oxidase reagent ( $6\%$ tetramethyl-p-phenylenediamine dihydrochloride in DMSO) reacts with cytochrome c and oxygen to produce a colored product.
- Results:
- Positive: Development of a dark blue to purple color on the disk.
- Negative: No color change.
- Organisms:
- Positive: Micrococcus spp.
- Negative: Staphylococcus spp.
PYR Test (Pyrrolidonyl Arylamidase)
- Biochemical Mechanism: Detects the ability of the organism to produce L-pyrrolidonyl arylamidase. This enzyme hydrolyzes the substrate L-pyrrolidonyl-b-naphthylamide into L-pyrrolidone and b-naphthylamine. The addition of cinnamaldehyde reagent reacts with the b-naphthylamine.
- Results:
- Positive: Bright red/pink color change.
- Negative: No color change or yellow/orange tint.
- Organisms:
- Positive: Streptococcus pyogenes (Group A), Enterococcus spp.
- Negative: Streptococcus agalactiae (Group B).
Novobiocin Susceptibility
- Biochemical Mechanism: The antibiotic novobiocin inhibits the DNA gyrase enzyme, preventing DNA replication and protein synthesis.
- Results:
- Resistant: Zone of inhibition < 16 \text{ mm}.
- Susceptible: Zone of inhibition $\geq 16 \text{ mm}$ .
- Organisms:
- Resistant: Staphylococcus saprophyticus (specifically used for urine isolates).
- Susceptible: Staphylococcus epidermidis and other CoNS.
Optochin (Taxo P) Susceptibility
- Biochemical Mechanism: Ethylhydrocupreine hydrochloride (Optochin) selectively interferes with the ATP synthase enzyme and lyses the cell wall of susceptible organisms.
- Results:
- Susceptible: Zone of inhibition $\geq 14 \text{ mm}$ with a $6 \text{ mm}$ disk.
- Resistant: No zone or zone < 14 \text{ mm}.
- Organisms:
- Susceptible: Streptococcus pneumoniae.
- Resistant: Viridans group streptococci (e.g., S. mitis, S. mutans).
CAMP Test
- Biochemical Mechanism: Identifies Group B Streptococci via the production of the "CAMP factor." This extracellular protein acts synergistically with the beta-lysin produced by S. aureus, resulting in enhanced lysis of sheep red blood cells.
- Results:
- Positive: Formation of a distinct "arrowhead" or wedge-shaped zone of beta-hemolysis where the two streaks meet.
- Negative: No enhancement of hemolysis.
- Organisms:
- Positive: Streptococcus agalactiae (Group B).
- Negative: Streptococcus pyogenes (Group A).
Bile Esculin Hydrolysis
- Biochemical Mechanism: Tests for the ability of an organism to grow in the presence of $40\%$ bile and produce the enzyme esculinase. Esculinase hydrolyzes esculin into esculetin and glucose. Esculetin then reacts with ferric citrate in the medium to form a phenolic iron complex.
- Results:
- Positive: Blackened medium.
- Negative: No color change.
- Organisms:
- Positive: Enterococcus spp., Streptococcus bovis group (Group D).
- Negative: Viridans streptococci.
6.5% NaCl Tolerance
- Biochemical Mechanism: Measures the ability of the organism to maintain osmotic balance and metabolic activity in extreme high-salt environments.
- Results:
- Positive: Visible growth (turbidity) in the broth.
- Negative: No growth (clear broth).
- Organisms:
- Positive: Enterococcus spp.
- Negative: Streptococcus bovis group (Group D).
Hippurate Hydrolysis
- Biochemical Mechanism: The enzyme hippuricase hydrolyzes sodium hippurate to benzoate and the amino acid glycine. Ninhydrin reagent is added to detect the presence of glycine through oxidative deamination.
- Results:
- Positive: Deep purple color.
- Negative: No color change or pale yellow.
- Organisms:
- Positive: Streptococcus agalactiae.
- Negative: Streptococcus pyogenes.
LAP (Leucine Aminopeptidase) Test
- Biochemical Mechanism: Detects the enzyme aminopeptidase, which hydrolyzes the substrate L-leucine-b-naphthylamide to release b-naphthylamine, which is then visualized using cinnamaldehyde reagent.
- Results:
- Positive: Red color change.
- Negative: No color (yellow).
- Organisms:
- Positive: Streptococcus spp., Enterococcus spp.
- Negative: Leuconostoc spp.