8.3 - Nonfermentative Bacilli

What are the most appropriate screening tests to presumptively differentiate and identify the nonfermentative gram-negative bacilli (NFB) from the Enterobacteriaceae species?

A. Catalase, decarboxylation of arginine, growth on blood agar

B. Motility, urease, morphology on blood agar

C. Oxidase, TSI, nitrate reduction, growth on MacConkey agar

D. Oxidase, indole, and growth on blood agar

C. Oxidase, TSI, nitrate reduction, growth on MacConkey agar

NFB vs. Enterobacteriaceae differentiation

• Oxidase: NFB are oxidase positive (most), Enterobacteriaceae are oxidase negative.

• TSI: NFB show no acid (K/K) → nonfermenters; Enterobacteriaceae ferment glucose/lactose/sucrose.

• Nitrate reduction: variable among NFB; most Enterobacteriaceae reduce nitrate → nitrite.

• MacConkey growth: both grow, but NFB appear as non–lactose fermenters (colorless colonies).

Presumptive tests used for identification of the Pseudomonas spp are:

A. Oxidase, oxidation-fermentation (OF) glucose (open), OF glucose (sealed), motility, pigment production

B. Growth on blood agar plate (BAP) and eosin-methylene blue (EMB) agars, lysine decarboxylation, catalase

C. Growth on MacConkey, EMB, and XLD agars and motility

D. Growth on mannitol salt agar and flagellar stain

A. Oxidase, oxidation-fermentation (OF) glucose (open), OF glucose (sealed), motility, pigment production

Pseudomonas identification

• Oxidase positive, motile, and aerobic (oxidative) in the OF test (acid only in open tube).

• Pigment production (e.g., P. aeruginosa → pyocyanin, fluorescein) supports ID.

• Nonfermenter, grows well on BAP and MacConkey (NLF).

• Confirms genus-level ID among nonfermentative Gram-negative bacilli (NFB).

Which tests are most appropriate to differentiate between Pseudomonas aeruginosa and Pseudomonas putida?

A. Oxidase, motility, pyoverdin

B. Oxidase, motility, lactose

C. Oxidase, ONPG, DNase

D. Mannitol, nitrate reduction, growth at 42C

D. Mannitol, nitrate reduction, growth at 42C

P. aeruginosa vs P. putida differentiation

• P. aeruginosa grows at 42 °C, reduces nitrate to nitrogen gas, and is mannitol negative.

• P. putida does not grow at 42 °C, does not reduce nitrate to gas, and is typically mannitol positive.

• Growth temperature and nitrate reduction are the key distinguishing traits within the fluorescent pseudomonads.

Which test group best differentiates Acinetobacter spp from P. aeruginosa?

A. Oxidase, motility, nitrate reduction

B. MacConkey growth, 37C growth

C. Blood agar growth, oxidase, catalase

D. Oxidase, TSI, MacConkey growth

A. Oxidase, motility, nitrate reduction

Acinetobacter vs. Pseudomonas differentiation

Acinetobacter spp: oxidase negative, nonmotile, and do not reduce nitrate.

• Pseudomonas aeruginosa: oxidase positive, motile (polar flagella), and reduces nitrate → nitrogen gas.

• Both are nonfermentative Gram-negative bacilli, but these three tests provide a rapid presumptive distinction.

In addition to motility, which test best differentiates Acinetobacter spp and Alcaligenes faecalis?

A. Triple sugar iron agar

B. Oxidase

C. Urease

D. Flagellar stain

B. Oxidase

Acinetobacter vs. Alcaligenes differentiation

• Acinetobacter spp: oxidase negative, nonmotile, often coccobacillary.

• Alcaligenes faecalis: oxidase positive, motile, with a fruity odor and alkaline TSI (K/K).

• Oxidase testing, combined with motility, provides a rapid and reliable separation of these two nonfermenters.

The most noted differences between P. aeruginosa and Stenotrophomonas maltophilia are:

A. Oxidase, catalase, and TSI

B. Oxidase, catalase, and ONPG

C. Oxidase, 42C growth, and polar tuft of flagella

D. Catalase, TSI, and pigment

C. Oxidase, 42C growth, and polar tuft of flagella

C. Oxidase, 42 °C growth, and polar tuft of flagella
P. aeruginosa vs. S. maltophilia (

• P. aeruginosa: oxidase positive, grows at 42 °C, and has a polar tuft of flagella; often produces pyocyanin pigment.

• S. maltophilia: oxidase negative, no growth at 42 °C, and has peritrichous flagella; typically DNase +, yellow-green colonies.

• These traits provide a rapid, reliable distinction between the two nonfermentative Gram-negative rods.

Which nonfermentative bacillus is usually associated with a lung infection related to cystic fibrosis (CF)?

A. Pseudomonas fluorescens

B. Pseudomonas aeruginosa

C. Pseudomonas putida

D. Burkholderia pseudomallei

B. Pseudomonas aeruginosa

Cystic fibrosis pathogen

• P. aeruginosa is the most common NFB causing chronic lung infections in CF patients.

• Forms mucoid biofilms due to alginate capsule, leading to persistent colonization and resistance.

• Key traits: oxidase +, growth at 42 °C, grape-like odor, and blue-green pigment (pyocyanin).

• Major cause of morbidity and mortality in CF.

A nonfermenter recovered from an eye wound is oxidase positive, motile with polar monotrichous flagella, and grows at 42C. Colonies are dry, wrinkled or smooth, buff to light brown, and are difficult to remove from the agar. In which DNA homologous group should this organism be placed?

A. Pseudomonas stutzeri

B. Pseudomonas fluorescens

C. Pseudomonas putida

D. Burkholderia pseudomallei

A. Pseudomonas stutzeri

DNA group identification

• P. stutzeri (DNA homology group I) shows oxidase positive, motile (polar flagella), growth at 42 °C, and dry, wrinkled, adherent colonies (tan to buff).

• Often recovered from wound and environmental sources.

• Differentiated from:

  • P. fluorescens / P. putida → no growth at 42 °C.

  • Burkholderia pseudomallei → typically smoother, non-adherent colonies and distinct clinical presentation (melioidosis).

Which organism is associated with immunodeficiency syndromes and melioidosis (a glanders-like disease prevalent in Southeast Asia and northern Australia)?

A. Pseudomonas aeruginosa

B. Pseudomonas stutzeri

C. Pseudomonas putida

D. Burkholderia pseudomallei

D. Burkholderia pseudomallei

Melioidosis pathogen

• Causes melioidosis, a glanders-like disease endemic to Southeast Asia and northern Australia.

• Found in soil and water; infection via inhalation, ingestion, or percutaneous inoculation.

• Opportunistic, often in immunocompromised or diabetic patients.

• Gram-negative, oxidase positive, motile bacillus; resembles Pseudomonas but is biochemically inert and nonpigmented.

Which characteristics/biochemical tests are used to differentiate Burkholderia cepacia from S. maltophilia?

A. Pigment on blood agar, oxidase, DNase

B. Pigment on MacConkey agar, flagellar stain motility

C. Glucose, maltose, lysine decarboxylase

D. Triple-sugar iron agar, motility, oxidase

A. Pigment on blood agar, oxidase, DNase

Burkholderia cepacia vs. Stenotrophomonas maltophilia

• B. cepacia: oxidase positive (variable), DNase negative, may produce yellow-green pigment on blood agar.

• S. maltophilia: oxidase negative, DNase positive, produces lavender-green pigment.

• Both are nonfermenters associated with respiratory infections, especially in cystic fibrosis patients.

The following results were obtained from a pure culture of gram-negative rods recovered from the pulmonary secretions of a 10-year-old cystic fibrosis patient with pneumonia:

Oxidase +

Glucose OF (open) +

Pigment: Red

Motility +

Gelatin hydrolysis +

Arginine dihydrolase (nonfluoresence) +

Growth at 42C: +

Flagella + (polar, monotrichous)

Which is the most likely organism?

A. Burkholderia pseudomallei

B. Pseudomonas stutzeri

C. Burkholderia cepacia

D. Pseudomonas aeruginosa

D. Pseudomonas aeruginosa

Rationale

• Grows at 42 °C, oxidase +, motile (polar, monotrichous), arginine dihydrolase +.

• Produces pigments, including red pyorubin (in addition to pyocyanin/pyoverdin variants).

• Classic CF respiratory pathogen; contrasts with B. cepacia (usually no 42 °C growth), P. stutzeri (dry, wrinkled buff colonies), B. pseudomallei (different clinical/biochem profile).

Alcaligenes faecalis (formerly A. odorans) is distinguished from Bordetella bronchiseptica with which test?

A. Urease (rapid)

B. Oxidase

C. Growth on MacConkey agar

D. Motility

A. Urease (rapid)

Alcaligenes faecalis vs. Bordetella bronchiseptica

• B. bronchiseptica is rapid urease positive (within minutes).

• A. faecalis is urease negative or weak/delayed positive.

• Both are oxidase positive, motile, and grow on MacConkey agar, so the urease reaction is the key differentiator.

Chryseobacterium spp are easily distinguished from Acinetobacter spp by which of the following 2 tests?

A. Oxidase, growth on MacConkey agar

B. Oxidase and OF (glucose)

C. TSI and urea hydrolysis

D. TSI and VP

A. Oxidase, growth on MacConkey agar

Chryseobacterium vs. Acinetobacter

• Chryseobacterium spp. are oxidase positive and generally do not grow or grow poorly on MacConkey agar.

• Acinetobacter spp. are oxidase negative and grow well on MacConkey agar (often as NLF colonies).

• These two traits provide a rapid and reliable distinction between the genera.

A gram-negative coccobacillus was recovered on chocolate agar from the CSF of an immunosuppressed patient. The organism was nonmotile and positive for indophenol oxidase but failed to grow on MacConkey agar. The organism was highly susceptible to penicillin. The most probable identification is:

A. Acinetobacter spp

B. Pseudomonas aeruginosa

C. Pseudomonas stutzeri

D. Moraxella lacunata

D. Moraxella lacunata

Rationale

• Gram-negative coccobacillus, oxidase positive, nonmotile, no growth on MacConkey, and penicillin susceptible → consistent with Moraxella lacunata.

• Commonly causes ocular and CNS infections in immunocompromised patients.

• Differentiated from:

  • Acinetobacter: oxidase negative, grows on MacConkey.

  • P. aeruginosa/P. stutzeri: motile, grow on MacConkey, resistant to penicillin

Cetrimide agar is used as a selective isolation agar for which organism?

A. Acinetobacter spp

B. Pseudomonas aeruginosa

C. Moraxella spp

D. Stenotrophomonas maltophilia

B. Pseudomonas aeruginosa

Cetrimide agar use

• Cetrimide inhibits most bacteria except P. aeruginosa, making the medium selective.

• Stimulates production of pyocyanin and fluorescein pigments, which aid identification.

• Colonies often appear greenish with a grape-like odor.

• Used for confirmation and isolation of P. aeruginosa from clinical or environmental specimens.

A specimen from a 15 yo female burn patient was cultured after debridement, and the following results were obtained:

Oxidase +

Catalase +

Ornithine decarboxylase neg

Lysine decarboxylase neg

Motility +

Glucose + for oxidation (open tube)

Maltose neg for oxidation (open tube)

Arginine dihydrolase +

Penicillin: R

Aminoglycosides: S

Colistin: S

These results indicate which of the following organisms?

A. Acinetobacter spp

B. Moraxella lacunata

C. Pseudomonas aeruginosa

D. Alcaligenes spp

C. Pseudomonas aeruginosa

• Oxidase +, motile, oxidative glucose (open tube) with arginine dihydrolase + → classic P. aeruginosa profile.

• Maltose (oxidation) −, Lysine/ornithine decarb − further support ID.

• Susceptibility pattern: Penicillin R, aminoglycosides S, colistin S fits P. aeruginosa.

• Clinical fit: common burn wound pathogen.

A yellow pigment-producing organism, growing on chocolate agar, testing oxidase positive, nonmotile and does not grow on MacConkey agar was recovered from the blood of a neonate. What is the most likely organism?

A. Acinetobacter spp

B. Pseudomonas aeruginosa

C. Burkholderia cepacia

D. Elizabethkingia (formerly Chryseobacterium) meningosepticum

D. Elizabethkingia (formerly Chryseobacterium) meningosepticum

Rationale

• Produces a yellow pigment, is oxidase positive, nonmotile, and fails to grow on MacConkey agar.

• Known cause of neonatal meningitis and sepsis, especially in premature or immunocompromised infants.

• Differentiates from:

  • Acinetobacter: oxidase negative, grows on MacConkey.

  • Pseudomonas: motile, grows on MacConkey, no yellow pigment.

  • Burkholderia cepacia: motile and grows on MacConkey.

Which reagent(s) is (are) used to develop the red color indicative of a positive reaction in the nitrate reduction test?

A. Sulfanilic acid and alpha-naphthylamine

B. Ehrlich and Kovac reagents

C. o-Nitrophenyl-beta-D-galactopyranoside

D. Kovac reagent

A. Sulfanilic acid and alpha-naphthylamine

Nitrate reduction test interpretation

• Purpose: Detects bacterial ability to reduce nitrate (NO₃⁻) to nitrite (NO₂⁻).

• Reagents: Sulfanilic acid + α-naphthylamine react with nitrite to produce a red azo dye (positive).

• No color after reagents: Add zinc dust—if red develops, nitrate was unreduced (true negative); if still colorless, nitrate reduced beyond nitrite to N₂ gas (true positive).

• Used for: Differentiating Enterobacteriaceae and nonfermenters (e.g., Pseudomonas spp., Neisseria spp.).

A culture from an intra-abdominal abscess produced orange-tan colonies on blood agar that gave the following results:

Oxidase +

KIA: Alk/Alk (H2S)+

DNase +

Growth at 42C: Neg

Nitrate reduction +

Motility: + (single polar flagellum)

Ornithine decarboxylase +

MacConkey agar: NLF

The most likely identification is:

A. Shewanella putrefaciens

B. Acinetobacter spp

C. Pseudomonas aeruginosa

D. Chryseobacterium spp

A. Shewanella putrefaciens

Key features for ID

• Nonfermenter (KIA Alk/Alk) with H₂S+ → hallmark of Shewanella among oxidase-positive GNRs.

• Oxidase+, motile (single polar flagellum), NLF on MAC, nitrate+ → matches S. putrefaciens.

• Grows poorly at 42 °C (negative) → differentiates from P. aeruginosa.

• DNase+ and orange-tan colonies consistent with reported Shewanella phenotypes.

Differentiates from

• P. aeruginosa: usually grows at 42 °C, H₂S−, often produces pyocyanin.

• Acinetobacter spp: oxidase−, many are nonmotile, often ONPG+.

• Chryseobacterium spp (Elizabethkingia): pigment-producing but nonmotile and H₂S−.

Chryseobacterium spp and B. cepacia are easily differentiated by which test?

A. Motility

B. OF glucose

C. Oxidase

D. Cetrimide agar

A. Motility

Chryseobacterium vs. Burkholderia differentiation

• Chryseobacterium spp.: nonmotile, oxidase positive, yellow pigmented, no growth on MacConkey or cetrimide.

• Burkholderia cepacia: motile (polar flagella), also oxidase positive, grows on MacConkey, no pigment.

• Thus, motility is the most reliable and rapid differentiating test.

Which of the listed Pseudomonas spp is associated with the following virulence factors: exotoxins A, endotoxins, proteolytic enzymes, antimicrobial resistance, and production of alginate?

A. Pseudomonas fluorescens

B. Pseudomonas putida

C. Pseudomonas stutzeri

D. Pseudomonas aeruginosa

D. Pseudomonas aeruginosa

Key distinguishing features of Pseudomonas aeruginosa

• Produces exotoxin A, endotoxin, proteases, and alginate (biofilm matrix).

• Highly drug resistant via efflux pumps and biofilm protection.

• Grows at 42 °C, oxidase positive, motile, and forms blue-green pigment on Cetrimide agar.

• Causes burn wound, CF, and nosocomial infections.

Summary
P. aeruginosa is distinguished by its toxin production, alginate biofilm, and multidrug resistance.

A 20 yo horse groomer exhibited a "glanders-like" infection. His history indicated he had suffered several open wounds on his hands 2 weeks before the swelling of his lymph nodes. A GNR was recovered from a blood culture that grew well on blood & Mac agars. Most of the biochemical tests were neg, including the cytochrome oxidase test. What is the most likely ID?

A. Burkholderia mallei

B. Pseudomonas aeruginosa

C. Pseudomonas stutzeri

D. Burkholderia pseudomallei

A. Burkholderia mallei

Key distinguishing features of Burkholderia mallei

• Causes glanders, a zoonotic infection primarily in horses and handlers.

• Nonmotile, oxidase negative, and grows on blood and MacConkey agar.

• Biochemically inert with most reactions negative.

• B. pseudomallei is motile and oxidase positive, differentiating it from B. mallei.

• Pseudomonas spp unlikely — both P. aeruginosa and P. stutzeri are oxidase positive, motile, and typically cause environmental or nosocomial infections, not zoonoses.

Summary
B. mallei is the nonmotile, oxidase-negative agent of glanders, acquired from horses and producing localized or systemic abscesses.

A Vietnam war veteran presented with a "glanders-like" infection (melioidosis). Several blood cultures produced GNRs that were positive for cytochrome oxidase, oxidized glucose and Xylose, and grew at 42C. What is the most likely organism?

A. Stenotrophomonas maltophilia

B. Burkholderia pseudomallei

C. Pseudomonas aeruginosa

D. Acinetobacter spp

B. Burkholderia pseudomallei

Key Distinguishing Features of Burkholderia pseudomallei

• Causes melioidosis, a glanders-like infection seen in veterans or travelers from Southeast Asia.

• Motile, oxidase positive, and oxidizes glucose and xylose.

• Grows at 42 °C, unlike B. mallei.

• Wrinkled colonies with earthy odor; may resemble Pseudomonas on culture.

• B. mallei is nonmotile and oxidase negative, differentiating the two.

• Pseudomonas aeruginosa is oxidase positive but typically does not oxidize xylose and produces pyocyanin pigment.

Summary
B. pseudomallei is a motile, oxidase-positive, 42 °C-growing organism that causes melioidosis, a chronic glanders-like disease endemic to Southeast Asia.

Cytochrome oxidase-positive nonfermentative GNB were recovered from the stool of a patient with CF. The isolates produced wet (mucoidy) light blue colonies on tryptic soy agar. Which identification is most likely?

A. Acinetobacter spp

B. Pseudomonas putida

C. Pseudomonas stutzeri

D. Pseudomonas aeruginosa

D. Pseudomonas aeruginosa

Key Distinguishing Features of Pseudomonas aeruginosa

• Source: Frequently isolated from cystic fibrosis (CF) patients; colonizes respiratory or GI tract.

• Colony morphology: Mucoid, wet, blue-green colonies from pyocyanin and alginate production.

• Biochemical traits: Oxidase positive, nonfermentative, motile, grows at 42 °C.

• Differentiation: P. putida and P. stutzeri are nonmucoid, less virulent, and fail to grow at 42 °C.

Summary
P. aeruginosa uniquely forms mucoid blue-green colonies and grows at 42 °C, distinguishing it as the CF-associated pathogen.

Several postoperative hospitalized patients were colonized with GNCB growing on Mac agar. Specimens were obtained from blood, urine, and wound sites. Testing revealed oxidase negative, nonmotile organisms. Which of the following is the most likely cause of the nosocomial infections?

A. Acinetobacter spp

B. Pseudomonas aeruginosa

C. Burkholderia cepacia

D. Pseudomonas putida

A. Acinetobacter spp

Key Distinguishing Features of Acinetobacter spp

• Clinical setting: Frequent nosocomial pathogen in postoperative, ventilator, and urinary infections.

• Morphology: Gram-negative coccobacilli (GNCB), sometimes appearing coccoid on Gram stain.

• Growth: On blood and MacConkey agar as non–lactose fermenters.

• Biochemical traits: Oxidase negative, nonmotile, catalase positive.

• Differentiation: Unlike Pseudomonas and Burkholderia, which are oxidase positive and motile.

Summary
Acinetobacter spp are oxidase-negative, nonmotile GNCB that cause hospital-acquired wound and device infections.

A nosocomial infection involving an 80-year-old female patient, recovering from pneumonia, produced many oxidase-negative colonies on MacConkey agar. Further testing results are:

Motility +

Maltose +

Resistant to Beta-Lactams

Glucose +

Resistant to most aminoglycosides

The most likely identification is:

A. Acinetobacter spp

B. Stenotrophomonas maltophilia

C. Pseudomonas aeruginosa

D. Burkholderia gladioli

B. Stenotrophomonas maltophilia

Key Distinguishing Features of Stenotrophomonas maltophilia

• Clinical niche: Nosocomial colonizer/pathogen in post-pneumonia, ventilated, or debilitated patients.

• Growth/appearance: Oxidase negative NLF on MacConkey; aerobic nonfermenter.

• Motility/biochem: Motile (polar flagella); maltose oxidative +; glucose oxidative +.

• Resistance pattern: Intrinsically resistant to many β-lactams and most aminoglycosides; classically susceptible to TMP-SMX.

• Differentiation: Unlike Acinetobacter (nonmotile), P. aeruginosa (oxidase +), and Burkholderia (different sugar oxidation profile and resistance pattern).

Summary
Profile of an oxidase-negative, motile, maltose-positive nonfermenter with broad β-lactam and aminoglycoside resistance in a hospital setting → consistent with Stenotrophomonas maltophilia.