Lab 9 Quiz - Non-fermenters + Campylobacter

Common Specimen Location of Pseudomonas aeruginosa

Sputum and other LRT specimens - especially CF patients

What is a non-fermenter

Aerobic g-b that cannot utilize glucose, or does so oxidatively

Examples of non-fermenters

Psuedomonas, Stenotrophomonas, Acinetobacter, Alcaligenes, Flavobacterium

How to differentiate Nonfermenters from Enterobacteriaceae

- MAC growth - white, or no colonies

- Positive cytochrome oxidase test on 18-24H BAP

- TSI: No change, or K/K (no glucose fermentation)

Most common infections of nonfermenters

Opportunistic agents of nosocomial infections - potent medications, instrumentation, prolonged surgeries

Clinical screening tests for non-fermenters

Oxidase (pos), OF test (oxidative or non-saccharolytic), Motility for genera

Clinical determination of non-fermenters

API, MALDI-TOF

Associated diseases of Psuedomonas aeruginosa

- Seen in CF patients

- Hot tube syndrome (folliculitis),

- Cellulitis

- Otitis externa (swimmers ear)

- Eye infection (bacterial keratitis)

Psuedomonas BAP

Irregular shaped, metallic sheen pigment

- Grape-like odor from colonies (walmart flip-flops)

Testing for Pseudomonas

- Oxidase positive

- TSI K/K

- Growth at 42C

- Motility pos (wet prep)

- Nitrate reduction (denitrification)

- Pigment production

Pigments produced by Pseudomonas aeruginosa

Fluorescein (pyoverdin), Pyocyanin, Pyorubin, Pyomelanin

What compound makes P.aeruginosa fluoresce?

Fluorescein (pyoverdin) - view BAP in short wave UV light

What makes Pseudomonas aeruginosa blue?

Pyocyanin

Why does P.aeruginosa have a metallic sheen?

Pyocyanin. On a MAC it may look blue

Wet prep motility

Add a drop of TSB to coverslip, examine right away under 10X and 40X

Positive result for wet prep motility

Organisms dart/tumbling across field in one direction

Negative result for wet prep motility

Organisms only have brownian movement (jerky, oscillatory movement inn random directions)

Bacteria glucose metabolism

Some bacteria can metabolize and produce acid only under aerobic conditions, while others can metabolize in aerobic and anaerobic conditions

Aerobic glucose metabolism (oxidation)

Glucose is oxidized to gluconic acid (a weak acid), which can undergo further oxidation

Anaerobic glucose metabolism (fermentation)

Glucose is phosphorylated and split into two triose molecules. The triose molecules can create lactic, formic, succinic, or acetic acid

Why is fermentation media visible quicker than oxidation

No oxygen required, so larger amounts of acid are producd.

Example of non-motile organism

Klebsiella sp.

Example of motile organism

Psuedomonas aeruginosa

Use of the OF test

Determines if sugar media is attacked by oxidation or fermentation

OF test medium

High carbohydrate content (glucose), low peptones, 0.3% agar, phosphate, NaCL, bromothymol blue

How to run OF test

Inoculate two tubes with media. One tube will have the cap left open (oxidative), and the other will be sealed with mineral oil and have the cap lid screwed on tightly (fermentative). Incubate 24H and read results the next day

Do we mix OF tubes?

No - can introduce oxygen to sample. Just stab media

OF test results for oxidative organism

Aerobic tube turns yellow, anaerobic tube is green

OF test results for fermentative organism

Aerobic tube turns yellow, anaerobic tube turns yellow

OF test results for non-saccharolytic organism

Aerobic tube stays green, anaerobic tube stays green

Example of fermentative organism (OF test)

Escherichia coli

Example of oxidative organism (OF test)

Psuedomonas aeruginosa

Unique result of Psuedomonas OF test

Aerobic tube turns yellow on top of media - Psuedomonas is a strict aerobe

Theory of OF test

Organism is inoculated onto sugar media with bromothymol blue indicator. If the organism metabolizes carbohydrates, acid is produced (Aerobic = gluconic acid. Anaerobic = formic, lactic, succinic, acetic acid). Acid production lowers the pH of media, and the bromothymol blue changes from green to yellow

Nitrate reduction test

Test to see if an organism can reduce nitrates to nitrites (or further reduced) using nitrate reductase

Nitrate reductase principle

- Nitrate reductase converts nitrate into nitrite

- Nitrite reacts with acetic acid to form nitrous acid

- Nitrous acid reacts with sulfanilic acid (reagent A) to form diazolium salt

- The diazolium salt reacts with dimethyl-alpha-napthylamide (reagent B) to form a red-colored azo dye (p-Sulfobenzene-azo-napthylamine)

Denitrification

In nitrate reductase test, organisms reduce nitrate past nitrite, into ammonia, nitrogen, nitric/nitrous oxide, or a hydroxylamine. In the test, addition of reagents will give negative result.

In a nitrate reduction test, the test tubes remained a clear color after addition of reagents. What is the next step?

Add zinc dust to the tubes. Zinc will reduce any nitrate left over. After zinc addition:

- If the tube turns red, the organism is negative (the zinc reduced the nitrate to nitrite)

- If the tube stays white, the organism is positive (there is no nitrate for zinc to reduce, as all the nitrate was denitrified)

Example of a nitrate reductase positive organism?

Escherichia coli

Example of nitrate reductase positive organism (with denitrification) ?

Psuedomonas aeruginosa

Example of nitrate reductase negative organism

Acinetobacter baumanii

Uses of nitrate reductase test

- Differentiate between Enterobacteriaceae (all pos, some denitrify)

- Differentiates Mycobacterium

- Identifies Neisseria sp from Moraxella and Kingella

- Differentiate Neisseria gonorrhoeae from Kingella denitrificans, as they have similar gram stain and colony morph

- Helps ID Corynebacterium sp

- Nitrate reduction can be linked to anaerobic respiration in some species

Colony morphology of Psuedomonas aeruginosa

Lggy sheen

What 3 tests are susepct of non-fermenters?

- White/no growth on MAC (with growth on BAP)

- TSI is K/K

- Positive cytochrome oxidase reaction

Campylobacter jejuni colony morph

Clear, flat and spreading, or drop-like

Campylobacter jejuni gram stain

G-b, curved "gull wing" colonies

Campylobacter jejuni motility

Corkscrew/darting motility (wet prep)

Campylobacter jejuni oxidase result

Positive

Campylobacter jejuni catalase result

Positive

Campylobacter jejuni growing conditions

42 degree celsius incubation for 48 hours in microaerophillic conditions (5-10% O2, 3-10% CO2)

Campylobacter jejuni pathogenicity

Causes human diarrheal disease (esp. children). Symptoms are abdominal cramps, profuse watery diarrhea containing mucus, blood, and bile

Workup of Psuedomonas aeruginosa

Colony morph: Lggy sheen (BAP), Wht or none (MAC)

Gram stain: G-b

Oxidase: pos

TSI: K/K

OF Test: Oxidative

Nitrate reductase: Pos (denitrification)

Fluorescein pigment in UV light