Enterobacteriaceae and Pseudomonas: Identification and Differentiation in the Clinical Laboratory

Enterobacteriaceae and Pseudomonas

Background and Clinical Importance

The Enterobacteriaceae ("enterics") are a large family of Gram-negative rods that inhabit the intestines of humans and animals. They include both normal flora and important human pathogens. Pseudomonas is a genus of Gram-negative rods, primarily free-living in soil and water, with Pseudomonas aeruginosa being a notable opportunistic pathogen. Differentiation and identification of these bacteria are crucial in clinical microbiology for diagnosis and treatment of infections.

• Gram-negative rods: Appear pink/red after Gram staining due to their thin peptidoglycan layer and outer membrane.

• Lactose fermentation: Used to distinguish between normal flora and pathogens among enterics.

• All enterics ferment glucose, but only some ferment lactose.

Normal Flora vs. Pathogenic Enterics

• Normal flora (lactose positive): Escherichia coli

• Human pathogens (lactose negative): Salmonella spp., Shigella spp., Vibrio cholerae

Pathogenic enterics can cause severe infections, including gastroenteritis, dysentery, and septicemia.

Major Pathogenic Enterics

• Salmonella: Causes typhoid fever, gastroenteritis, and septicemia. Transmission often occurs via contaminated water, food (especially eggs), or reptiles.

• Shigella: Infects humans and primates, causing symptoms from mild diarrhea to severe bacillary dysentery.

• Vibrio: Vibrio cholerae causes cholera, characterized by "rice water" stools and severe dehydration. Vibrio parahaemolyticus is associated with seafood-borne gastroenteritis.

Isolation and Differentiation of Enteric Bacteria

Selective and Differential Media

Selective and differential media are essential tools for isolating and identifying enteric bacteria from clinical specimens.

EMB (Eosin Methylene Blue) Agar

• Selective for Gram-negative rods: Dyes inhibit Gram-positive bacteria.

• Differential for lactose fermentation:

  • Lactose fermenters (e.g., E. coli) produce colonies with a green metallic sheen.

  • Other lactose fermenters (e.g., Enterobacter aerogenes) show pink colonies with dark centers.

  • Non-lactose fermenters produce clear or colorless colonies.

MacConkey Agar

• Selective for Gram-negative rods: Bile salts and crystal violet inhibit Gram-positive bacteria.

• Differential for lactose fermentation:

  • Lactose fermenters form red/purple colonies (e.g., E. coli forms dark red colonies).

  • Non-lactose fermenters form colorless or pale colonies.

Colony Morphology on Selective Media

• Lactose fermenters: Pink/red colonies on MacConkey; green sheen or dark centers on EMB.

• Lactose non-fermenters: Colorless or pale colonies on MacConkey; clear colonies on EMB.

Biochemical Identification of Enteric Bacteria

Triple Sugar Iron (TSI) Agar Test

The TSI test differentiates bacteria based on their ability to ferment glucose, lactose, and sucrose, produce gas, and generate hydrogen sulfide (H2S).

• Indicator: Phenol red (detects acid production).

• Iron compounds: Detect H2S production (blackening of medium).

• Interpretation:

  • Yellow butt/red slant: Glucose fermentation only.

  • Yellow butt/yellow slant: Lactose and/or sucrose fermentation.

  • Red butt/red slant: No sugar fermentation.

  • Gas production: Splitting or bubbles in agar.

  • Black color: H2S production.

IMViC Tests

The IMViC series (Indole, Methyl Red, Voges-Proskauer, Citrate) is a set of biochemical tests used to distinguish among enteric bacteria, especially E. coli and Enterobacter species.

• Indole Test: Detects the ability to produce indole from tryptophan. Positive result: red color after adding Kovac's reagent.

• Methyl Red Test: Identifies mixed acid fermentation. Positive result: red color at pH 4.

• Voges-Proskauer Test: Detects butanediol fermentation (acetoin production). Positive result: red color after adding reagents.

• Citrate Utilization Test: Determines ability to use citrate as sole carbon source. Positive result: growth and blue color change.

Summary Table: IMViC Test Results

Test	Positive Result	Negative Result
Indole	Red color after Kovac's reagent	No color change
Methyl Red	Red color (pH < 4)	Yellow color (pH > 6)
Voges-Proskauer	Red color after reagents	No color change
Citrate	Growth and blue color	No growth, green color

Enterotube System

The Enterotube is a multi-test system containing 12 different media, allowing for 15 standard biochemical tests from a single bacterial colony. It is used for rapid identification of enteric bacteria in clinical labs.

Pseudomonas

Background and Identification

• Gram-negative rods, primarily free-living in soil and water.

• Oxidase positive (distinguishes from enterics).

• Pseudomonas aeruginosa is a prominent opportunistic pathogen, resistant to many antibiotics and disinfectants.

• Can grow at high temperatures (up to 42°C), high salt, and in the presence of dyes and weak antiseptics.

Identification of Pseudomonas aeruginosa

• Oxidase test: Positive result is a black color within 20–30 minutes.

• Growth on Pseudosel agar (PSA): Contains cetrimide to inhibit other bacteria and stimulates production of pyocyanin (blue-green pigment) and fluorescein (fluorescent pigment).

• Mueller Hinton agar: Selective for P. aeruginosa; colonies appear green and may fluoresce under UV light.

• Glucose fermentation: Pseudomonas does not ferment glucose; colonies may have a fruity, grape-like odor.

Summary Table: Key Laboratory Tests for Enterics and Pseudomonas

Test	Enterobacteriaceae	Pseudomonas
Gram Stain	Negative rods	Negative rods
Oxidase	Negative	Positive
Lactose Fermentation	Variable	Negative
Glucose Fermentation	Positive	Negative
Growth on EMB/MacConkey	Yes	Poor/None
Pigment Production	None	Blue-green (pyocyanin)

Additional info: The above notes integrate foundational microbiology concepts with laboratory diagnostic techniques, providing a comprehensive overview for college-level study and exam preparation.