oxidase-negative

**Principle of the test:** Cytochrome c oxidase (complex IV of the electron transport chain) transfers electrons from cytochrome c to O2. In the lab, an artificial electron acceptor (tetramethyl-p-phenylenediamine, or dimethyl-p-phenylenediamine in the Gordon-McLeod reagent) is oxidized by the enzyme and turns dark blue/purple. Oxidase-positive organisms turn purple within about 10-30 seconds. Oxidase-negative organisms show no color change. Use a platinum, plastic or wooden applicator, not an iron-containing nichrome loop, which can give false positives.

**Oxidase-negative organisms (high-yield):** - Enterobacterales (all lactose fermenters and non-fermenters): *E. coli*, *Klebsiella*, *Enterobacter*, *Citrobacter*, *Serratia*, *Salmonella*, *Shigella*, *Proteus*, *Yersinia*, *Morganella*, *Providencia* - *Acinetobacter baumannii* (a non-fermenter; this separates it from *Pseudomonas*) - *Stenotrophomonas maltophilia* (variable or weak; usually reported as negative) - *Haemophilus* species are oxidase-positive, so don't confuse them. - Most gram-positives are not tested, because the test is mainly applied to gram-negatives. *Staphylococcus* and *Streptococcus* are catalase-based comparisons instead.

**Oxidase-positive organisms (mnemonic for contrast):** *Pseudomonas aeruginosa*, *Neisseria*, *Moraxella*, *Vibrio*, *Campylobacter*, *Helicobacter*, *Legionella*, *Aeromonas*, *Pasteurella*, *Haemophilus*, *Brucella* (variable), *Bordetella pertussis*.

**Clinical and diagnostic significance:** - The oxidase test is a quick first step to split gram-negative rods into Enterobacterales (oxidase-negative, glucose fermenters) and non-fermenting or other oxidase-positive organisms. - Most oxidase-negative Enterobacterales ferment glucose, reduce nitrate to nitrite, and grow on MacConkey agar. Lactose fermenters (*E. coli*, *Klebsiella*, *Enterobacter*) appear pink. Non-lactose fermenters (*Salmonella*, *Shigella*, *Proteus*, *Yersinia*) appear colorless. - Pseudomonas vs. Acinetobacter: both are non-fermenting, aerobic, gram-negative organisms associated with hospital-acquired infection (ventilator-associated pneumonia, burns, line infections). *Pseudomonas* is oxidase-positive and *Acinetobacter* is oxidase-negative. - Clinical relevance: oxidase-negative Enterobacterales cause UTI (*E. coli*, the most common cause), pneumonia and liver abscess (*Klebsiella*), gastroenteritis and enteric fever (*Salmonella*), dysentery (*Shigella*), and struvite stones (*Proteus*). Carbapenem-resistant Enterobacterales (CRE) and ESBL producers are important oxidase-negative resistant pathogens.

**Exam pearls:** - Oxidase-negative + lactose fermenter + pink on MacConkey → *E. coli*, *Klebsiella*, *Enterobacter*. - Oxidase-negative + non-lactose fermenter + H2S positive → *Salmonella* or *Proteus*. Both produce H2S. *Proteus* is also urease-positive and swarms on agar. - A non-fermenting gram-negative rod that is oxidase-negative should make you think *Acinetobacter*. If it is oxidase-positive with a blue-green pigment and grape-like odor, think *Pseudomonas aeruginosa*. - Oxidase activity is related to the electron transport chain. Organisms with cytochrome c oxidase use O2 as the terminal electron acceptor in respiration.