Non-fermenting, G(-) Baccili

• Non-fermentative Gram-Negative Bacilli

  • Pseudomonadaceae
  • Vibrionaceae
  • Campylobacteraceae
  • Helicobacteraceae
• This is a flow chart which shows how we can easily differentiate gram-negative bacilli since this is a large group of organisms mainly populated by Enterobacteriaceae. We can easily differentiate them by performing oxidase test.

  
• Oxidase negative organisms

  1. Test further in the O/F medium

  2. If they are positive for oxidizing carbohydrates as well as fermenting carbohydrates – largely, organisms with this type of reactions are Enterobacteriaceae
• Oxidase positive organisms

  1. Test using the O/F media

  2. Those who show variable reactions to oxidation and fermentation using the O/F media might be Pseudomonadaceae or Vibrionaceae
• Pseudomonads

  Positive for oxidation, but they cannot ferment carbohydrates
• Vibrionaceae


  Positive for both oxidation and fermentation. Their reaction to O/F media are similar to Enterobacteriaceae, but of course, they are oxidase positive
• Characteristics of Non-Fermenters

  • Non-spore formers
  • Obligate aerobes
  • TSI: K/K and H2S (-)
  • Point of Colonization: Mucus membrane and skin of humans
  • Entry to sterile sites of the body: Trauma, burns, or wounds
  • Opportunistic pathogens
• A non-fermenter is suspected if the reactions of the laboratory test come up as; Oxidase positive Gram-Negative bacilli (GNB), Grow better in SBA than on MacConkey agar (+/-), TSI – K/K
• To further differentiate or identify non-fermenters

  1. Tests;

  2. Flagellar morphology

  3. Modified Indole Test

  4. Growth on MacConkey

  5. Motility

  6. O-F CHO utilization patterns

  7. Nitrate reduction

  8. Acetamide utilization test

  9. Urease

  10. Decarboxylase Reactions

  
• Oxidation-Fermentation (O-F) Test: Hugh & Leifson

  • Purpose: To determine whether an organism can utilize CHO oxidatively or fermentatively
  • High concentration of CHO (1%) and a low concentration of peptone or protein (0.2%)
  • 1:5 peptone to CHO concentration
  • Composition: OF Basal media, CHO: Glucose, Maltose, Lactose, Mannitol, and Xylose, Bromthymol blue = indicator

  
• Oxidative-Fermentation Reaction

  • Alcaligenes faecalis: Open Tube Green / Blue Green, Closed Tube Green / Blue Green, Non-saccharolytic
  • Pseudomonas: Open Tube Yellow, Closed Tube Green / Blue Green, Oxidative
  • Enterobacteriaceae: Open Tube Yellow, Closed Tube Yellow, Fermentative
• Nitrate Reduction Test

  1. Nitrate broth is used to demonstrate the ability of organisms to reduce nitrate to nitrite

  2. Add Sulfanilic acid and a-naphthylamine -> if a red color develops, it indicates a positive result for nitrate reduction to nitrite

  3. If no red color, add zinc dust - red color = nitrate not reduced, no color = nitrate was consumed

  
• Nitrate Reduction Test Tubes

  • Tube 1: No change with A-naphthylamine and Sulfanilic acid, Nitrite absent
  • Tube 2: Turns red with a-naphthylamine and sulfanilic acid, Nitrite present, positive nitrate reduction
  • Tube 3: No change with zinc, Positive nitrate reduction test
  • Tube 4: Turns red with zinc, Negative nitrate reduction test

  
• Family Pseudomonadaceae (NFGNB)

  • I. Fluorescent Group: Pseudomonas aeruginosa, P. fluorescens, P. putida

  Non-Fluorescent Group (Soil denitrifiers): P. stutzeri, P. mendocina

  II. Pseudomallei Group: Burkholderia pseudomallei, B. mallei, B. cepacia, Ralstonia pickettii

  III. Comamonas spp, Acidovorax spp

  IV. Brevundimonas spp

  V. Stenotrophomonas maltophilia
• Pseudomonas aeruginosa (PAE)


  Not a major normal flora, but most frequently isolated N-F, accounts for about 75 – 80% of non-fermenting gram-negative bacteria isolated from clinical specimens
• P. stutzeri and P. mendocina


  Capable of denitrifying the soil
• Burkholderia pseudomallei group

  • Burkholderia pseudomallei
  • B. mallei
  • B. cepacia
  • Ralstonia picketii
• Burkholderia pseudomallei group


  • Known for their characteristic resistance to polymyxin B and Colistin
• Comamonas spp, Acidovorax spp, and Brevundimonas spp have not been isolated as much as other NFGNB in human infections
• Group V


  • Stenotrophomonas maltophila
• Stenotrophomonas maltophila


  Shares resistance to polymyxin B and colistin with Group III, hence often grouped under Group III together with Burkholderia spp, Pseudomallei group
• Pseudomonas aeruginosa (PAE)


  Not a major normal flora but most frequently isolated non-fermenting gram-negative bacteria from clinical specimens, accounting for 75-80%
• Pseudomonas aeruginosa

  • Opportunistic pathogen
  • Can be identified by pigments it produces: pyocyanin (blue), pyoverdine (green), fluorescent (yellow), brown, and red
• Pseudomonas aeruginosa is found in the environment and can survive in harsh environments
• Pseudomonas aeruginosa


  • Major human pathogen in the group
  • Most important opportunistic pathogen
  • Invasive and toxigenic
• Pseudomonas aeruginosa

  • Obligate aerobe
  • Motile with polar flagellum
  • Produces pigments: pyoverdine, fluorescein, pyocyanin, pyomelanin, pyorubrin
• Pseudomonas aeruginosa can produce 2-aminoacetophenone which gives it a fruity or corn tortilla-like odor
• Pseudomonas aeruginosa

  • Grows at room temperature, 35-37 C, and 42 C
  • Ability to grow at 42 C separates it from other fluorescent Pseudomonas species
• Specimen best cultured or processed for identification of Pseudomonas aeruginosa

  • Skin lesions
  • Pus
  • Urine
  • Blood
  • Spinal fluid
  • Sputum
• Pseudomonas aeruginosa colonies on BAP

  • Large, rough, dull grayish, feathered edged/serrated and spreading, mostly beta-hemolytic, mucoid (capsular alginate)
• Pseudomonas aeruginosa on MacConkey and EMB

  • Non-lactose fermenting
• Biochemical tests for Pseudomonas aeruginosa

  • TSI: K/K
  • Oxidase Test: Positive
  • Oxidative Fermentation: Oxidizes glucose, fructose, xylose but not maltose, sucrose, lactose
  • Nitrate Reduction Test: Positive
• Other lab tests for Pseudomonas aeruginosa

  • MacConkey: NLF
  • TSI: K/K
  • Pyocyanin and Pyoverdine: Positive
  • Oxidase: Positive
  • O-F medium: (see above table)
  • LOA: Negative, Negative, Positive
  • Acetamide Utilization: Positive
  • Cetrimide test: Positive
  • Nitrate reduction: Positive
  • Citrate: Positive
  • Urease: Variable
  • Indole: Negative
  • Growth at 42°C: Positive
  • Motility: Motile with one flagella
  • Kanamycin: Resistant
  • Carbenicillin: Susceptible
• Infections associated with Pseudomonas aeruginosa

  • Nosocomial/HAI: Severe wound infections in burn patients, UTI, ventilator-associated pneumonia, septicemia, chronic lung infections in cystic fibrosis, meningitis, ecthyma gangrenosum
  • Community-acquired: Keratitis, corneal ulcers in contact lens wearers, swimmer's ear, folliculitis ("hot tub folliculitis")
• Pseudomonas aeruginosa virulence factors

  • Attachment: Fimbriae, polar flagella
  • Lipopolysaccharide endotoxin effect and exotoxin: Alginate capsule, Exotoxin A, Type III secretion system, Exotoxin S, T, U, Y
  • Tissue Invasion: Elastase, Protease, Collagenase, Hemolysin, Leukocidin, Pyocyanin, Pyoverdin
• Pseudomonas aeruginosa treatment

  • Single drug therapy not recommended, combination therapy with beta-lactam and aminoglycoside preferred
  • Beta-lactams, carbapenems, and fluoroquinolones can be used
  • Antibiotic susceptibility testing is important before treatment
• P. fluorescens and P. putida

  Fluorescent pseudomonads that can produce pyoverdine but not pyocyanin
• P. fluorescens and P. putida

  • Found in the environment
  • Can grow at 4°C
  • Rarely associated with opportunistic infections
• P. fluorescens

  Contaminant that can cause food spoilage and rare cause of UTI and wound infections
• P. putida

  Isolated cases of septicemia, UTI, and pneumonia, found in hospital environments especially around sinks