NONFERMENTING AND MISCELLANEOUS GRAM NEGATIVE BACILLI

• General Characteristics of Non-Fermenters

  • Fail to acidify an oxidative-fermentative (OF) medium
  • Fail to acidify triple sugar iron agar (TSIA) butts
  • Grow in aerobic environment
  • Some members oxidize carbohydrates, others are non-oxidizers or asaccharolytic
  • Oxidase positive
  • All species except Burkholderia mallei are motile, having one or several polar flagella
  • Optimum temperature range: 30° to 37° C (mesophilic)
  • Acidovorax facilis is MacConkey negative
  • Pseudomonas spp. are catalase positive
• Carbohydrate utilization in Triple Sugar Iron Agar (TSIA)

  • Slanted portion used for lactose and sucrose fermentation
  • Butt portion used for glucose fermentation
  • Non fermenters remain alkaline [K/K] [red slant/red butt] - will not ferment the sugars
• Biochemical Characteristics and Identification of Non-Fermenters
• Biochemical Characteristics and Identification of Non-Fermenters

  • Oxidase-positive reaction, although reaction can be weak and variable
  • Non-reactivity in 24 hours in commercial multi-test kit systems used primarily for the identification of enterobacteriaceae
  • No acid production in the slant or butt of TSIA or KIA
  • Resistance to a variety of classes of antimicrobial agents, such as aminoglycosides, third-generation cephalosporins, penicillin, and fluoroquinolones
• Different Classification Systems for the Non-Fermenters

  • Growth on MAC Agar
  • Oxidase reaction
  • Glucose O-F Test
• Pseudomonas

  • Gram-negative bacillus or coccobacillus
  • Strictly aerobic metabolism
  • Motile usually with polar flagellum or polar tuft of flagella
  • Oxidase positive (except P. luteolus and P. oryzihabitans)
  • Catalase positive
  • Usually grows on MAC agar
  • Usually an oxidizer of carbohydrates, but some species are asaccharolytic
  • Produce water-soluble pigments, yellow green pigments (pyoverdine), blue pigment (pyocycanin)

  
• Pseudomonas Fluorescent Group

  • Pseudomonas aeruginosa
  • Pseudomonas fluorescens
  • Pseudomonas putida
  • Pseudomonas monteilii
  • Pseudomonas veronii
  • Pseudomonas mosselii
• Pseudomonas Non-Fluorescent Group

  • Pseudomonas stutzeri
  • Pseudomonas mendocina
  • Pseudomonas alcaligenes
  • Pseudomonas pseudoalcaligenes
  • Pseudomonas luteola
  • Pseudomonas oryzihabitans
• Pseudomonas Aeruginosa


  • Motile and rod shaped
  • Obligate aerobe
  • Producing a sweet or grape-like or corn taco-like odor = 2 Aminoacetophenone
  • Produce a non-fluorescent bluish pigment, pyocyanin
  • Produces the fluorescent pigment pyoverdine
  • May also produce a dark red pigment (pyorubin) or a brown black pigment (pyomelanin)
• Growth Characteristics of Pseudomonas Aeruginosa

  • Grows well at 37°C to 42°C - 42°C can differentiate them from other pseudomonas species
  • Does not ferment carbohydrates
  • Many strains oxidize glucose - oxidase positive
  • Denitrification of nitrates and nitrites
  • Arginine dihydrolase (ADH) -positive
  • Citrate positivity
  • Acetamide utilization
  • Grows well on Cetrimide Agar - a selective and differential medium for identification

  
• Cetrimide Agar


  • Acts as a detergent inhibiting most of the bacteria
  • Enhances the production of the pigments
• Exotoxin A


  Blocks the protein synthesis
• Clinical Findings of Pseudomonas Aeruginosa

  • Wounds and burns - blue-green pus
  • Necrotizing pneumonia
  • Cystic fibrosis - chronic pneumonia
  • Mild otitis externa in swimmers
  • Invasive (malignant) otitis externa in patients with diabetes
  • Fatal sepsis - infants
  • Ecthyma gangrenosum - presence of erythema or redness without pus
  • Folliculitis - poorly chlorinated hot tubs and swimming pools
• Treatment of Pseudomonas Aeruginosa

  • Piperacillin and ticarcillin
  • Third- and fourth generation cephalosporins (ceftazidime and cefepime, respectively)
  • Carbapenems (except ertapenem)
  • Fluoroquinolones
• Pseudomonas Fluorescens and Pseudomonas Putida

  • Can be isolated from respiratory specimen, contaminated blood products even on urine and other hospital equipment
  • Can grow at 4°C
  • Produce pyoverdine
  • Cannot reduce nitrate to nitrogen gas
  • Can produce acid from xylose
  • P. putida: Gelatin hydrolysis negative
  • P. fluorescens: Gelatin hydrolysis positive

  
• Pseudomonas Mosselii


  • Non-pigmented and non-hemolytic
  • Positive for oxidase, catalase, and ADH
  • Optimal growth occurs at 30°C

  
• Pseudomonas Stutzeri


  • Wrinkled, leathery, adherent colonies
  • Light-yellow or brown pigment
  • ADH negative
  • Starch hydrolysis positive
  • Can grow in an anaerobic environment in nitrate containing media—able to produce nitrogen gas
  • Causes septicemia, meningitis, pneumonia, endocarditis, postsurgical wound infection, septic arthritis, conjunctivitis, and UTIs
• Pseudomonas Mendocina


  • Produces non-wrinkled, flat colonies that may appear with a yellowish-brown pigment
  • Many exhibit a smooth buttery appearance
  • Oxidase and ADH positive
  • Does not produce pyoverdine
  • Acetamide negative
  • Motile - single polar flagellum
  • Oxidizes glucose and xylose
  • Nonproteolytic and does not hydrolyze starch

  
• Pseudomonas Pseudoalcaligenes and Pseudomonas Alcaligenes

  • Oxidase positive
  • Biochemically negative in may tests
  • Grow on MAC agar
  • Motile - polar flagellum
  • P. pseudoalcaligenes - ADH positive and will weakly ferment fructose
  • Often considered as contaminants if isolated from clinical specimens

  
• Pseudomonas Luteola and Pseudomonas Oryzihabitans

  • Gram-negative, non-fermentative, oxidase-negative bacilli
  • Catalase positive and motile, oxidize glucose, grow on MAC agar
  • Produce an intracellular non-diffusible yellow pigment
  • Produce wrinkled or rough colonies at 48 hours
  • P. luteola can be differentiated from P. oryzihabitans by the o-nitrophenyl-B-D-galactopyranoside (ONPG) test and esculin hydrolysis

  
• Acinetobacter


  • Aerobic, catalase-positive, oxidase negative, Gram-negative bacteria
  • May also appear as diplococci on smears and then resemble Neisseria
  • Grow best at 35°C to 37°C

  
• Acinetobacter Baumannii


  • Most commonly isolated in clinical laboratories
  • Glucose-oxidizing, nonhemolytic strain

  
• Acinetobacter Radioresistens


  • Described to colonize human skin and cause occasionally infections in immunocompromised patients
• Acinetobacter Iwoffi


  • Glucose negative
  • Nonhemolytic strain
• Clinical Infections of Acinetobacter

  • Primarily affect patients with weakened immune systems and coexisting diseases
  • UTIs; pneumonia, tracheobronchitis, or both; endocarditis
  • Septicemia; meningitis
  • Cellulitis - usually from contamination of catheters or from introduction of a foreign body
  • A. baumannii - eye infections: endophthalmitis, conjunctivitis, and corneal ulcerations
• Identifying Characteristics of Acinetobacter

  • Can resist decolorization and retain the crystal violet stain
  • Purplish hue produced by some species
  • A. baumannil is saccharolytic, and A. woffii is asaccharolytic
  • CRAB (carbapenem resistant A. baumanni) - only susceptible to colistin and tigecycline
• Stenotrophomonas Maltophilia


  • Third most common non-fermentative, gram-negative bacillus in the clinical laboratory
  • Colonies may appear bluish on MAC agar
  • Positive for catalase, DNase, esculin and gelatin hydrolysis, and lysine decarboxylase

  
• Burkholderia Cepacia Complex


  • Opportunistic organism
  • Weak, slow, positive oxidase reaction causes onion bulb rot in plants and foot rot in humans motile by means of polar tufts of flagella ornithine decarboxylase negative and fail to reduce nitrate to nitrite lusine decarboxylase and ONPG positive oxidize glucose and many will oxidize maltose, lactose, mannitol
• Burkholderia Mallei


  • Causes infections
• Acinetobacter


  • Can resist decolorization and retain the crystal violet stain
  • Produces a purplish hue
  • A. baumannii is saccharolytic, A. woffii is asaccharolytic
  • CRAB (carbapenem resistant A. baumanni) is only susceptible to colistin and tigecycline
• Stenotrophomonas maltophilia


  • Third most common non-fermentative, gram-negative bacillus in the clinical laboratory
  • Colonies may appear bluish on MAC agar
  • Positive for catalase, DNase, esculin and gelatin hydrolysis, and lysine decarboxylase

  
• Burkholderiacepacia Complex


  • Opportunistic organism
  • Weak, slow, positive oxidase reaction
  • Oxidize glucose, and many will oxidize maltose, lactose, and mannitol
  • Lysine decarboxylase and ONPG positive
  • Ornithine decarboxylase negative and fail to reduce nitrate to nitrite
• Burkholderia mallei


  • Respiratory tract zoonosis
  • Old name was Pseudomonas mallei
  • Smooth and cream to white colonies in BAP and weakly oxidase (+)
  • The only non-motile among all pseudomonads
  • Can't grow in 42°C
  • Causes Glander's disease - Infectious disease of horses, goats, sheep, & donkey. Rare cause of human infection acquired by direct contact, trauma, or inhalation
  • Farcy - disseminated form of glander's disease
  • Considered as a potential bioterrorism agent

  
• Burkholderia cepacia complex

  • Burkholderia cepacia
  • Burkholderia mallei
• Burkholderia pseudomallei


  • Cream to tan wrinkled colonies in BAP
  • Motile via polar tuft flagella - ADH (+)
  • Highly oxidative - glucose, maltose, lactose, mannitol
  • Causes Melioidosis - glanders-like disease which has a long latent period - A pulmonary disease (transmitted via ingestion, inhalation, or inoculation of organism etc.)
  • Manifestation of symptoms takes years hence the synonym Vietnam Time Bomb
  • Produces yellow pigment after 48-72 hours of incubation
• Burkholderia gladioli


  • Motile by means of one or two polar flagella
  • Catalase and urease positive
  • Grows on MAC agar
  • Oxidizes glucose
  • Mannitol positive and decarboxylase negative, and is negative for oxidase, although some strains are weakly positive
• Alcaligenes and Achromobacter


  • Can be found on water even on swimming pools
  • Resistant to disinfectants
  • Obligately aerobic gram-negative bacilli
  • Possess peritrichous flagella
  • May produce a fruity odor
  • Cause a green discoloration on SBA
  • OF media: nonoxidative, produce a deep blue color at the top, except for A. xylosoxidans (old name of achromobacter xylosoxidans var. denitrificans)

  
• Brevundimonas

  • B. diminuta: Motile and possess a single polar flagellum, oxidize glucose, oxidase positive, Most strains grow on MAC agar
  • B. vesicularis: Slender rod, with polar flagella, Only about 25% grows on MAC agar, Most strains produce an orange intracellular pigment, Oxidase positive and oxidizes glucose and maltose

  
• CDC Groups E0-3, E0-4, and Paracoccus


  • Oxidase-positive
  • Nonmotile, saccharolytic coccobacili that grow weakly, if at all, on MAC agar
  • All oxidize glucose and xylose
  • EO-3 and many EO-4 isolates have a yellow non-diffusible pigment
  • P. yeei (EO-2) coccoid or O shaped cells on Gram stain
• Chromobacterium violaceum


  • Motile, facultative anaerobe
  • Oxidase (+)
  • Rare cause of human infection
  • Found in soil and water
  • Unique because of the violet pigment that it produces Violacein