Parvo & Misce

• Miscellaneous group

  Heterogenous small gram-negative coccobacilli, they are non-motile, non-spore-forming, aerobic, and microaerophilic bacteria
• Genera in the miscellaneous group

  • Haemophilus
  • Bordatella
  • Brucella
  • Francisella
• Haemophilus

  Gram-negative, tiny coccobacilli, pleomorphic, similar in ultrastructural features to other pathogenic bacilli
• Haemophilus influenzae

  Requires hemin (factor X), and NAD (factor V) for growth
• Other Haemophilus species

  Only require NAD, and therefore grow on blood agar

  
• Typable H. influenzae isolates

  Classified on the basis of seven antigenically distinct polysaccharides

  
• Non-typable H. influenzae

  Isolates lacking the seven polysaccharides
• Haemophilus

  • Non-motile, non-spore-forming, facultative anaerobic
  • Most species are oxidase and catalase positive
  • Preferred incubation: 5 – 10% CO2 @ 35 – 37*C
  • Species are very prone to drying, and chilling
  • Most species are non-pathogenic except for H. influenzae, H. ducreyi, and H. aegyptius

  
• Haemophilus influenzae


  The major pathogen which can be separated into encapsulated or typable strains, and unencapsulated or non-typable strains

  
• Type B H. influenzae


  The most virulent organism, commonly causing blood stream invasion and meningitis in children younger than 2 years
• Non-typable H. influenzae strains


  Frequent causes of respiratory tract disease in infants, children, and adults
• Other Haemophilus species

  • H. parainfluenzae sometimes cause pneumonia and bacterial endocarditis
  • H. ducreyi causes chancroid
  • H. aprophilus is a member of the normal flora of the mouth and occasionally causes bacterial endocarditis
  • H. aegyptius causes conjunctivitis and Brazilian purpuric fever
  • H. haemolyticus

  
• Haemophilus species constitute 10% of the normal flora of the upper respiratory tract
• Haemophilus

  The term "Haemophilus" means "Blood-loving" in Greek
• Haemophilus cell wall

  Contains lipooligosaccharide, which resembles the lipolysaccharide of gram-negative bacilli, but has shorter side chains
• Haemophilus species have generally been thought not to make toxins or other extracellular products that account of their ability to produce infection

  
• X factor


  The only readily available factor required by Haemophilus

  
• V factor


  Not readily available and requires exogenous sources
• Haemophilus

  Requires hemin or factor X, and Nicotinamide Adenine Dinucleotide (NAD) or factor V for growth
• NAD

  Released into the medium by the red blood cells and is available to the bacteria in the blood agar
• Hemin


  Bound to red blood cells and it is not released in to the medium unless the cells are broken up as in a chocolate agar
• Haemophilus isolation

  • Special growth requirements for identification
  • Requires X factor (degradation product of hemoglobin, heat-stable, also known as hemin/hematin)
  • Requires V factor/NAD/Coenzyme-1 (produced by some bacteria, yeast, potato and yeast extract, heat labile)

  
• 1. ALA test (Delta-Aminolevulenic Acid Test)/ Porphyrin Test


  Also known as ALA porphyrin Test, used to detect the requirement of X-factor
• ALA negative

  X factor dependent organism
• ALA positive

  H. parainfluenzae, can convert ALA to porphobilinogen, and porphyrin to Hemin
• H. influenzae

  ALA negative, needs exogenous X factor, positive under UV light – it produces a red fluorescence at 360 nm, which indicates the presence of porphyrin

  
• Sheep blood agar


  Not suitable for Haemophilus, lacks NAD required by the organism for growth
• Sheep RBCs

  Release NADase enzyme except when grown with Staphylococcus
• Horse or rabbit blood agar


  Ideal culture media as it has no NADase enzyme
• Chocolate agar

  The preferred isolation medium, heated at 80*C to release hematin (X-factor) and inactivate NADase
• Levinthal agar or Levinthal chocolate agar


  Nutrient agar added with 5% horse blood with X and V factors
• Encapsulated H. influenzae

  Produce iridescent colonies
• Non-encapsulated H. influenzae

  Transparent, bluish, and iridescent in appearance of their colonies
• Synthetic chocolate agar

  Contains NAD, Iron, Vit B12, thiamine, magnesium, glucose, cysteine, and glutamine
• Satellitism


  Phenomenon in which H. influenzae forms dew drop colonies in the vicinity of Staphylococcal colonies, a distinguishing factor of H. influenzae
• Horse blood-bacitracin agar

  Beef-heart infusion, peptone, yeast, 5% defibrinated horse blood (provides both X and V factor), 300 mg/L Bacitracin (inhibits normal flora)
• Use of X and V factors

  For speciation of Haemophilus, using Mueller-Hinton agar with X, V, & XV filter paper disks

  
• Haemophilus influenzae

  Also known as Pfeiffer's bacillus, divided into encapsulate and non-encapsulated strains

  
• Haemophilus influenzae

  • Gram-negative coccobacilli, smooth drop-like colonies on CAP, require X and V factor on the nutrient agar, show satellitism near SAU colonies, and show pleomorphism from clinical specimens

  
• H. influenzae capsule

  Made of polysaccharide, antigenic, increases pathogenicity, and prevents phagocytosis