non fermentive

• Nonfermentative gram-negative bacilli


  They are opportunistic pathogens not usually found in the human body, usually found in the environment (water, soil and vegetables) and medical devices (nebulizers, ventilators, catheters, dialysate fluid and other hospital instruments)
• Nonfermentative bacilli

  • Non-lactose on Mac Konkey Agar
  • Exhibits alkaline slant and alkaline butt (K/K) in TSI (no acid production even in anerobic portion of the medium)
• Nonfermentative bacilli

  This group of organisms is differentiated from Enterobacteriaceae in the way they utilize carbohydrates, they do not ferment sugars (in the absence of air) but utilize oxidatively producing tiny amount of acid
• This group includes

  • Pseudomonas
  • Acinetobacter
  • Stenotrophomonas
  • Burkholderia
  • Alcaligenes

  
• Hugh and Leifson


  Realized that the organisms utilizing CHO minimally produce tiny amounts of acids, the conventional fermentation medium contains high content of peptone (1%) which these organisms utilize preferentially, producing enough alkaline amines to neutralize the small amounts of acids produced, thereby no pH reaction is seen
• OF Medium


  Hugh and Leifson developed low peptone medium (0.2%) to detect acid production of these organisms
• Purpose of OF Medium

  Used to determine if an organism can utilize carbohydrates in an oxidative or fermentative manner, and to identify those bacteria that are asaccharolytic, that is, unable to use carbohydrates

  
• Principle of OF Medium

  The medium used to identify Enterobacteriaceae is not suitable for nonfermenters as the amount of acid produced during the oxidative process is not sufficient for the indicator to take on its acidic color, and the alkaline end products produced during the reaction neutralize the acid produced

  
• Principle of OF Medium

  In OF medium, positive reactions are indicated by yellow color, as the Bromthymol blue indicator becomes yellow in an acidic environment. Green or blue-green is indicated as negative.
• Principle of OF Medium

  OF medium contains high concentration of carbohydrate (1%) and small concentration of peptone (0.2%), which facilitates the oxidative use of carbohydrates by nonfermenting gram-negative bacilli
• Procedure for OF Medium

  1. Inoculate top portion of the medium of 2 O-F tubes (for each carbohydrate tested) with growth taken from colonies

  2. Overlay the surface of one tube with ¼ inch of sterile mineral oil

  3. Replace caps of both tubes

  4. Incubate at 35° for 18-24 hours. Interpret reactions at 24 hours. If no change has occurred, re-incubate for an additional 18-24 hours
• Interpretation of OF Medium

  • Fermentative - organisms that able to ferment glucose (yellow) in the close tubes and oxidize it in the open tubes
  • Oxidative - organisms that give a yellow reaction only in the open tube indicating oxidative utilization of glucose
  • Non-oxidizer (non-saccharolytic) - do not utilize glucose neither fermentatively nor oxidatively (Blue-green)

  
• Pseudomonas aeruginosa


  • Aerobic, motile with single polar flagellum which has good growth at 42°C but pigment is best produced at room temperature
  • Gram (-) rods that occur singly, in pairs, and occasionally in chains
  • Produces extracellular slime layer, similar to a capsule usually seen in mucoid strains isolated from patients with cystic fibrosis
  • Frequently possesses pili that promote attachment to host cell surface

  
• Virulence Factors of Pseudomonas aeruginosa


  • Presence of glycocalyx
  • Pili/fimbriae - for adherence
  • Endotoxin
  • Extracellular enzymes - proteases, hemolysins, etc
  • Exotoxin A - causes tissue necrosis
  • Exotoxin S - play a role in necrotic injury
  • Cytotoxin and enterotoxin
• Cultural Characteristics of Pseudomonas aeruginosa

  • Organisms can be readily cultured on NA, BAP, MaC and Cetrimide agar plate
  • Grow well on 37-42°C, its growth in 42°C helps differentiate it from other Pseudomonas spp.
  • On BAP - large, flat, β-hemolytic colonies with a feathered edge and ground glass appearance
  • Colonies tend to spread giving off a characteristic odor resembling over-riped grapes (grape-like fruity odor or corn tortilla odor) which is due to a substance aminoacetophenone

  
• Pigments produced by Pseudomonas aeruginosa

  • Pyocyanin - blue, water-soluble, chloroform-extractable pigment
  • Pyoverdin (fluorescein) - yellow to green water-soluble pigment
  • Pyorubin - red
  • Pyomelanin - brown to black

  
• Diseases produced by Pseudomonas aeruginosa

  • Burn wound infections: traumatic and operative wound infections
  • Nosocomial infections like pneumonia (especially in cystic fibrosis patients), UTI, endocarditis, osteomyelitis, etc
  • Eye infections seen in contact lens wearer
  • Dermatologic infections
• Laboratory Identification of Pseudomonas aeruginosa

  • Flagellar stain/ Gram-stain
  • Culture: Selective media - Pseudosel agar, Media enhancing fluorescent pigment production: Pseudomonas F agar, GNF (Glucose N2- Fluorescein) agar, Flo agar
  • Biochemical Tests - TSI, Open OF (+) and close OF (-) utilizing glucose oxidatively, Oxidase and catalase (+), Unable to oxidize lactose, sucrose and maltose, Unable to decarboxylate lysine and ornithine, able to hydrolyze arginine
  • Serotyping of O antigen
  • Bacteriophage typing
  • Pyocin typing - for epidemiologic studies
• Treatment for Pseudomonas aeruginosa

  • Aminoglycosides (amikacin, gentamycin and tobramycin)
  • Extended spectrum penicillins (carbenicillin)
  • 3rd generation cephalosporin (ceftazimide and cefoperazone)

  
• Pseudomonas fluorescens/putida


  • Pseudomonas fluorescens and Pseudomonas putida are isolated from blood products, cosmetics, hospital equipment, urine and respiratory specimens
  • Associated with transfusion-associated septicemia
  • Can grow at 4°C but not in 42 °C

  
• Biochemical Test for Pseudomonas fluorescens/putida

  • TSI K/K
  • Motility (+)
  • Oxidase (+)
  • H2S (-)

  
• Acinetobacter


  • Organisms in this genus are short, rod-shaped to spherical, non-motile, oxidase-negative, strictly aerobic, and Gram-negative
  • In a Gram-stained smear they often appear in pairs and may be difficult to decolorize
  • Acinetobacter spp. are commonly found in soil and water and uncommonly found on the skin and mucous membranes of healthy people
  • Little is known about virulence factors in this group of organisms, but they do appear to form small

  
• cillins (carbenicillin)


  3rd generation cephalosporin (ceftazimide and cefoperazone)
• Pseudomonas fluorescens/putida


  • Isolated from blood products, cosmetics, hospital equipment, urine and respiratory specimens
  • Associated with transfusion-associated septicemia
  • Can grow at 4°C but not in 42 °C

  
• Biochemical Test

  1. TSI K/K

  2. Motility (+)

  3. Oxidase (+)

  4. H2S (-)
• Pseudomonas fluorescens


  • Nutrient agar plate
  • King's B medium plate
  • Under UV-visible light
• Acinetobacter


  • Short, rod-shaped to spherical, non-motile, oxidase-negative, strictly aerobic, and Gram-negative
  • Often appear in pairs and may be difficult to decolorize
  • Commonly found in soil and water and uncommonly found on the skin and mucous membranes of healthy people
  • Little is known about virulence factors but they do appear to form small amounts of endotoxin
  • Usually nonpathogenic, but have been increasingly associated with nosocomial septicemia, pneumonia, bacteriuria, and wound infection

  
• Laboratory Diagnosis of Acinetobacter

  1. Can be distinguished from pseudomonads by lack of motility, inability to reduce nitrates, and negative oxidase reaction

  2. May produce characteristic purplish colonies on MacConkey's agar

  3. Over 25 species, often lumped in Acinetobacter calcoaceticus–Acinetobacter baumanii complex

  4. Glucose oxidizing (saccharolytic) strains are usually A. baumanii; nonsaccharolytic strains are either A. lwoffi or A. haemolyticus

  
• Stenotrophomonas (Xanthomonas) maltophilia


  • Important cause of hospital-acquired infections in immunocompromised patients
  • Motile with multitrichous polar flagella
  • Colonies are rough, lavender green or gray, slightly α-hemolytic, with ammonia-like odor
  • Unable to grow in cetrimide
  • Oxidase, Ornithine decarboxylase (-)
  • Lysine decarboxylase (+)
  • Treated with Trimethoprim-sulfamethoxazole
• Burkholderia (Pseudomonas) cepacia


  • Commonly isolated from cystic fibrosis patients and has been isolated from endocarditis, septicemia, wound and urinary tract infections in immunocompromised patients
  • Colonies are yellow-green, with soil-like odor
  • Strains are motile and able to oxidize glucose, lactose, maltose, and mannitol
  • Lysine decarboxylase (+)
  • Resistant to polymyxin B and sensitive to chloramphenicol, cotrimoxazole and ceftazidime
• Burkholderia (Pseudomonas) mallei


  • Glander's bacillus
  • Causes Glander's – a disease of horses occasionally transmitted to humans by direct contact or through skin abrasions and inhalation of organisms
  • Small, non-motile, aerobic, pleomorphic, coccoid to rod-shaped organisms which are oxidase negative
  • Colonies on BHIA – grayish white, transluscent and later becoming yellowish and opaque
  • On MaC – Lactose negative
• Strauss Test


  Test used to diagnose glanders, wherein male guinea are inoculated intraperitoneally with suspected material; within 2-3 days the animal develops orchitis (tender and swollen scrotum)with purulent inflammation of the testes
• Treatment for Burkholderia (Pseudomonas) mallei

  Tetracycline and aminoglycoside
• Burkholderia (Pseudomonas) pseudomallei


  • Whitmore's bacillus - Causes Melioidosis – a glander's-like disease in humans transmitted via inhalation, ingestion, ingestion or skin puncture/abrasions
  • Four (4) forms of disease - Acute, subacute, chronic, latent which may produce abscesses and septicemia
  • Both chronic and latent may reactivate to a symptomatic form after many years thus named "VIETNAMESE TIME BOMB"
  • Small, motile with polar tufts of flagella, aerobic, gram-negative rods
  • Colonies are wrinkled at first becoming umbonate exhibiting varying degrees of hemolysis with a characteristic putrid odor followed by aromatic, pungent odor
• Treatment for Burkholderia (Pseudomonas) pseudomallei

  Cotrimoxazole, tetracycline, chloramphenicol
• Other Gram-negative Bacilli

  • Psychrobacter sanguinis
  • Alcaligenes faecalis
  • Oligella urethralis
  • Moraxella lacunata
  • Chromobacterium violaceum
  • Shewanella putrefaciens
• Alcaligenes faecalis


  • Obligate aerobe isolated in soil, water and hospital environments
  • Causes meningitis, endocarditis, bacteremia, UTI and lung and wound infection
  • Colonies on BAP have feathered edge, non-pigmented with fruity odor
  • On MaC - NLF
  • Motile by peritrichous flagella
  • Oxidase (+), Catalase (+), 6.5% NaCl (+), Nitrate reduction (-)
• Oligella


  • Aerobic coccobacilli
  • May colonize distal urethra causing serious and active infection
  • Colonies on BAP are small, white and opaque
  • No growth on MAC
  • Oxidase and catalase (+), asaccharolytic
  • Oligella urethralis – non-motile, oxidase (+), urease (-)
  • Oligella ureolytica – motile oxidase (+), urease (+)
• Moraxella lacunata


  • Morax-Axenfeld bacillus
  • Gram-negative coccobacilli
  • No growth on MaC
  • On BAP – small and pit the agar
  • Catalase (+), Oxidase (+), Nitrate reduction (+)
• Chromobacterium violaceum


  • Known for its violet pigmentation with "ammonia cyanide" odor
  • Facultative anaerobe, motile with polar flagella
  • Opportunistic pathogen that causes neutrophil deficiency in immunocompromised patients
  • On MaC – with visible growth at 42°C
  • On BAP – colonies are smooth with violet pigmentation (violacein pigment)
  • Catalase (+), Oxidase (v)