HACEK

• Organisms covered in this USM


  • Miscellaneous
  • Fastidious
  • Pleomorphic (many shapes)
  • Small, gram-negative bacilli
  • Require special nutrients for isolation and identification (ID)
• Organism classification - Family: Pasteurellaceae


  • Haemophilus
  • Actinobacillus
  • Pasteurella
  • Aggregatibacter

  
• Organism classification - HACEK group


  • Haemophilus
  • Aggregatibacter
  • Cardiobacterium
  • Eikenella
  • Kingella

  
• Other fastidious gram-negative bacilli

  • Capnocytophaga
  • Brucella
  • Francisella

  
• Haemophilus gram-stain appearance


  • Gram-negative pleomorphic coccobacilli or rods
  • Coccobacilli in direct smears
  • Rods with occasionally long filamentous rods from colony growth

  
• Haemophilus biochemical reactions


  Nonmotile, oxidase positive, catalase positive, nitrate reduction, ferments carbohydrates
• Haemophilus location in the body

  Obligate parasites on the mucous membranes

  
• Haemophilus species


  • H. influenzae
  • H. parainfluenzae
  • H. haemolyticus
  • H.parahaemolyticus
  • H. pittmaniae
  • H. aegyptius
  • H. ducreyi
  • H. segnis
  • H. aphrophilus
  • H. paraphrophilus

  
• Haemophilus definition


  • Greek word meaning "blood lover"
  • Organisms prefer growth factors present in blood
  • Require X and V factors
• X factor


  Hemin, hematin
• V (for Vitamin) factor

  Nicotinamide-adenine dinucleotide (NAD)
• Species with the prefix "Para"


  • Require only V factor
  • H. parainfluenzae produces X factor, requires V factor
• Hemolysis of 5% horse or rabbit blood agar

  • H. haemolyticus species and sometimes H. ducreyi
  • Sheep blood agar (SBA) only contains X factor and not V factor, some will not grow without V factor added because NADases present in blood agar plate (BAP) destroy V factor
  • Chocolate (CHOC) agar releases X and V factor and deactivates NADases
• Growth of fastidious organisms around other bacteria - Satellitism

  • S. aureus, S. pneumoniae, and Neisseria species that cause hemolysis release the factors or naturally produce V factor
  • Clinically significant exception—H. Ducreyi

  
• Haemophilus species constitute approximately 10% of normal flora of the upper respiratory tract in adults, 2% to 6% in children from birth through childhood, with a higher percentage colonization in daycare centers

  
• H. influenzae was erroneously named during the influenza worldwide pandemic (1889–1890), frequently isolated in infected patients with influenza and from postmortem lung cultures

  
• H. influenzae virulence factors

  • Capsule
  • Immunoglobulin A (IgA) proteases
  • Adherence by fimbriae and other structures
  • Outer membrane proteins and Lipopolysaccharide (LPS)

  
• H. influenzae capsule classification and serotypes

  • Possible antigenic distinct types—a, b, c, d, e, and f
  • Type is based on differences in the capsular polysaccharide
  • Serotype b (Hib) consists of unique polymer composed of ribose, ribitol, and phosphate, evidence suggests antiphagocytic and anticomplement
  • Hib vaccine is useful in reducing incidence of disease

  
• Nontypable H. influenzae (NTHi)


  • Some strains are not encapsulated (no capsule)
  • Referred to as nontypable strains
  • Invade the respiratory tract and tissues located around the same area
  • Cause localized infections

  
• H. influenzae IgA protease


  Cleaves IgA on mucous membranes

  
• H. influenzae adherence mechanisms


  • Not well defined
  • Most NTHi strains are adherent to human epithelial cells whereas most Hibs are not, may explain the tendency for NTHi strains to cause systemic infections

  
• H. influenzae outer membrane components


  • Not well defined
  • Antibody directed against antigen may play a significant role in immunity
• H. influenzae two patterns of disease

  • Invasive disease caused by encapsulated strains - septicemia, meningitis, arthritis, epiglottitis, tracheitis, pneumonia
  • Localized infection due to Hib vaccination by contiguous spread of NTHi - conjunctivitis, sinusitis, Otitis media with effusion (middle ear infection)

  
• Nontypable (non-encapsulated) strains cause lower respiratory tract infections primarily in older patients and individuals with underlying respiratory tract problems, including cystic fibrosis
• Haemophilus influenzae meningitis


  • Before development of a Hib vaccine, most cases occurred in children ages 3 months to 6 years
  • Bloodstream invasion and bacteremic spread follow colonization, invasion, and organism replication in the respiratory mucous membranes

  
• Haemophilus influenzae epiglottitis


  • Acute inflammation and swelling, causing airway obstruction
  • Affects children 2 to 4 years old
  • Requires emergency tracheostomy

  
• Haemophilus influenzae bacterial tracheitis


  • Life-threatening disease in young children
  • Arises after an acute, viral respiratory infection
  • Mild to moderate illness for 2 to 7 days that progresses rapidly
  • Use of broad-spectrum antibiotics imperative because thick secretions can occlude trachea
  • Disease must be differentiated from epiglottitis

  
• Haemophilus aegyptius and H. influenzae biogroup aegyptius

  Can cause conjunctivitis - acute, contagious, purulent conjunctivitis particularly in children

  
• Haemophilus influenzae biogroup aegyptius

  Can cause Brazilian purpuric fever (BPF) - occurs in warm tropical climates, recurrent conjunctivitis, high fever, vomiting, petechiae, purpura, septicemia, shock, and vascular collapse, high mortality, as high as 70% within 48 hours after onset

  
• Haemophilus ducreyi


  Causes sexually transmitted infection called genital ulcer disease (GUD) - chancroid, incubates 4 to 14 days, painful lesion with an irregular edge in genital and perianal areas, enlarged and draining lymph nodes (buboes)
• Haemophilus parainfluenzae


  Low incidence of pathogenicity, can cause otitis media, acute sinusitis, endocarditis (rare), may be a cause of some cases of pharyngitis in absence of other pathogens

  
• Specimen processing and isolation

  • Organisms known to die rapidly, plated within 10 minutes for maximum recovery
  • H. ducreyi - clean specimen site with sterile saline and sterile gauze, swab base of ulcer with cotton swab moistened with sterile phosphate-buffered saline, and plate immediately, can also aspirate pus from buboes
• Media selection


  • H. influenzae - CHOC agar with bacitracin (300 mg/L)
  • H. aegyptius - CHOC agar supplemented with 1% IsoVitaleX
  • H. ducreyi - GC agar (hemoglobin, fetal bovine serum [FBS], vancomycin to reduce genital flora), enriched chocolate, or Mueller-Hinton agar with 5% chocolatized lysed horse blood
• Growth requirements


  • For most species - incubate in a 5% to 10% CO2, 35–37° C, place culture media into an atmosphere with high humidity to prevent drying
  • For H. ducreyi - grow at 33° C at 5% to 10% carbon dioxide (CO2) for 7 days before reporting negative result
  • For H. aegyptius - same as for H. ducreyi except incubate for 4 days instead of 7
• Colony morphology of H. influenzae

  Translucent, tannish, moist, smooth, convex, mousy or bleach-like odor, encapsulated strains appear more mucoid

  
• Microscopic morphology of H. influenzae

  Gram: small gram-negative coccobacilli or small rods to long filaments, clear non staining areas (halos) may be seen

  
• Microscopic morphology of H. ducreyi

  Gram: coccobacilli arranged in groups resembling "school of fish," "railroad tracks," or "fingerprints"

  
• Laboratory ID initial findings

  Gram-negative pleomorphic coccobacilli on CHOC agar, no growth on SBA or on MacConkey (MAC) agar

  
• X and V Factor Requirement


  Take colonies from an initial isolation of Haemophilus, place the colonies in nutrient broth and mix, plate inoculated nutrient broth on a nutrient agar plate or a Mueller-Hinton agar plate, add X and V strips to the media, incubate at 35 to 37° C in 5% to 10% CO2 18 to 24 hours, read plates
• Organism requiring X and V factors

  H. influenzae