Enterobacteriales Flashcards

Characteristics of Enterobacteriales

Gram stain: Non-spore-forming Gram-Negative Bacilli (GNB)
Growth Characteristics: Facultative anaerobes
Colonies on SBA: Most are large, dull gray, non-hemolytic
Colonies on MAC:
- Lactose fermenters (pink)
- Non-lactose fermenters (colorless)
Biochemicals:
- Ferment glucose
- Oxidase negative
- Most reduce nitrates to nitrites
- Most are catalase positive

Principle: Detects acid products formed when glucose is metabolized via the mixed acid fermentation pathway.
Interpretation: A color change in a pH indicator signals the result.
Positive Result: Red color indicates a pH ≤ 4.5.
Comments: The test becomes positive at pH ≤ 4.5

Principle: Detects acetoin, an intermediate product from an alternate glucose metabolism pathway.
Process: Naphthol and KOH are added to the medium.
Positive Result: Red color develops after the addition of alpha-naphthol and KOH.
Comments:
- Little acid is produced via this pathway.
- Often inversely related to the MR test.
- Klebsiella and Enterobacter are typically VP positive.

Principle: Determines if an organism can use citrate as its sole carbon source, leading to a pH increase.
Interpretation: A pH indicator changes color if citrate is utilized.
Positive Result: Color changes from green to blue or growth on the medium.
Process: If the organism can use citrate as a sole source, the pH will increase, leading to a color change with a pH indicator.

Principle: Detects the breakdown of urea by urease, releasing ammonia, which increases pH.
Interpretation: pH indicator exhibits a color change.
Positive Result: Color changes from yellow to pink.
Comments: Proteae & Morganella are rapid urease producers.

Principle: PD deaminates phenylalanine to phenylpyruvic acid, which then reacts with ferric chloride.
Positive Result: A green color develops after adding ferric chloride.
Process: PD deaminates phenylalanine to phenylpyruvic acid. Positive = green color after adding ferric chloride.
Organisms: Proteus, Providencia, Morganella are positive for this test.

Principle: Detects the production of colorless H₂S gas from sulfur-containing compounds by organisms with appropriate enzymes.
Process: H₂S reacts with iron salt in the medium, forming black ferrous sulfide.
Positive Result: Black precipitate forms.
Comments:
- Sulfur-containing compounds: sodium thiosulfate, cysteine, methionine.
- Useful for differentiating Salmonella (H₂S positive) from Shigella (H₂S negative).

Principle: Detects the deamination of tryptophan by tryptophanase, producing indole.
Process: Kovacs reagent (paradimethylaminobenzaldehyde) forms a pink-colored complex with indole.
Positive Result: Pink color develops.
Process: Tryptophanase breaks down tryptophan to indole. Indole is detected using Kovacs reagent, forming a pink complex.
Organisms: E. coli and Proteus vulgaris are positive.

Principle: Detects sugar fermentation, which produces acid and is indicated by a pH indicator.
Colors: Yellow = acid (A), Pink = alkaline (K).
Reactions:
- Pink slant = glucose fermented.
- Yellow slant = lactose/sucrose not fermented.
- Yellow slant & butt = lactose and/or sucrose fermented.
- Black precipitate = H₂S produced.
- Bubbles = gas production.
Contents: Contains 0.1% glucose, 1% lactose, 1% sucrose, phenol red, sodium thiosulfate, iron salt.
Incubation: Leave air slightly loose.
Reporting: Record as slant/butt, e.g., K/A (alkaline slant/acid butt).
K/A (Same except no change) for non-fermenters.
A black butt indicates H₂S production.
Gas production is seen as bubbles or displacement of the agar.

Principle: Detects the presence of enzymes that remove a carboxyl group from amino acids (e.g., ornithine, lysine, arginine), leading to a pH increase.
Interpretation: pH indicator changes color.
Positive Result: Color changes from yellow to purple.
Process: Test for the presence of enzymes that remove a carboxyl group from amino acids. Positive result indicates the production of alkaline amines, raising pH.
Common Test: Ornithine decarboxylase (ODC).
Reactions:
- Most Enterobacteriales are positive, except Klebsiella & Shigella (negative).
- Differentiates Klebsiella & Enterobacter (positive) and differentiates Shigella (negative) & Salmonella (positive).

Principle: Determines if an organism can move away from the stab line in motility medium.
Positive Result: Movement away from stab line or hazy appearance throughout medium after overnight incubation.
Observation: Observed in semi-solid agar.
Motile bacteria will spread from the inoculation line, making the medium cloudy.
Non-motile bacteria will only grow along the stab line.

Principle: Detects the ability of an organism to ferment specific carbohydrates (e.g., lactose, sucrose, mannitol, etc.).
Process: When carbohydrate is fermented, acidic end products cause color change in pH indicator.
With phenol red indicator, color change from red to yellow.

Principle: Tests for the presence of the enzyme cytochrome c oxidase.
Process: Reagent changes color to blue or purple.
Positive Result: Blue or purple color develops.
Comments:
- Differentiates Enterobacteriales (oxidase negative) from other GNB (like Pseudomonas, which are oxidase positive).
- Avoid false positives by using a platinum loop or wooden stick instead of an iron-containing wire.

Principle: Determines if an organism can reduce nitrates to nitrites or further reduce nitrites to N₂ gas.
Process: Sulfanilic acid & alpha-naphthyl-amine are added.
Positive Result:
- Red color develops after adding reagents (nitrites are present).
- No color change after adding zinc dust (nitrites were reduced further to N₂).
Negative Result: Red color after adding zinc dust (nitrates are still present).
If color develops (red) after just adding reagents, nitrites are present.
If no color after reagents, add zinc dust.
If red develops after zinc dust, nitrates were still present (negative).
If no color after zinc dust and a gas bubble is present, nitrates were reduced to N₂ gas (positive).
All Enterobacteriales ferment glucose.

Principle: Detects the enzyme beta-galactosidase, which is required for lactose fermentation.
Process: ONPG is changed to ortho- nitrophenyl by Beta-galactosidase
Positive Result: Yellow color develops.
Detects slow lactose fermentation.
Helpful in differentiating Citrobacter (positive) from most Salmonella (negative).

Location: Capsule
Characteristics: Polysaccharide, heat labile, may mask O antigen. Removed by heating.
Role: Preventing phagocytosis.
Vi antigen is a K antigen produced by S. Typhi.