HACEK

• Haemophilus, HACEK, Legionella, and Other Fastidious Gram-Negative Bacilli
• Organisms covered


  • Miscellaneous
  • Fastidious
  • Pleomorphic (many shapes)
  • Small, gram-negative bacilli
  • Require special nutrients for isolation and identification
• 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 coccobacicili 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


  Obligate parasites on the mucous membranes
• Approximately 13 Haemophilus species known to exist, 8 species associated with humans
• Major pathogenic Haemophilus species

  • H. influenzae
  • H. aegyptius
  • H. ducreyi
• Haemophilus


  Greek word meaning "blood lover", organisms prefer growth factors present in blood, require X and V factors
• V factor


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


  Only require V factor, e.g. H. parainfluenzae
• Haemophilus haemolyticus species and sometimes H. decreyi cause hemolysis of 5% horse or rabbit blood agar
• Satellitism


  Growth of fastidious organisms around other bacteria that release the necessary growth factors or break down toxic products
• Haemophilus species constitutes approximately 10% of normal flora of the upper respiratory tract in adults, 2%-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)

  
• Virulence Factors of H. influenzae

  • Capsule
  • Immunoglobulin A (IgA) proteases
  • Adherence by fimbriae and other structures
  • Outer membrane proteins and lipopolysaccharide (LPS)
• Capsule serotype b (Hib)


  Consists of unique polymer composed of ribose, ribitol, and phosphate, evidence suggests antiphagocytic and anticomplement activity
• Nontypable H. influenzae (NTHi)

  Some strains are not encapsulated (no capsule), invade the respiratory tract and tissues located around the same area, cause localized infections
• Other Virulence Factors of H. influenzae

  • Immunoglobulin A (IgA) protease cleaves IgA on mucous membranes
  • Adherence mechanisms not well defined, most NTHi strains are adherent to human epithelial cells whereas most Hibs are not
  • Outer membrane components not well defined, antibody directed against antigen may play significant role in immunity
• Two Patterns of Disease caused by H. influenzae

  • Invasive disease caused by encapsulated strains (septicemia, meningitis, arthritis, epiglottitis, tracheitis, pneumonia)
  • More localized infection due to Hib vaccination by contiguous spread of NTHi (conjunctivitis, sinusitis, Otitis media with effusion)
• Clinical Manifestations of H. influenzae

  • Meningitis (before 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)
  • Epiglottitis (acute inflammation and swelling, causing airway obstruction, affects children 2 to 4 years old, emergency tracheostomy)
  • Bacterial Tracheitis (life-threatening disease in young children, arises after an acute, viral respiratory infection, thick secretions can occlude trachea, use of broad-spectrum antibiotics imperative)
• H. aegyptius and H. influenzae biogroup aegyptius

  Can cause conjunctivitis (pinkeye), particularly in children

  
• H. 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

  
• H. ducreyi


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


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

  
• Specimen Processing and Isolation

  1. Organisms known to die rapidly, plated within 10 minutes for maximum recovery

  2. 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 (300mg/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 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
• H. influenzae colony morphology

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

  
• H. influenzae microscopic morphology

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

  
• H. ducreyi microscopic morphology

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

  
• Laboratory ID Initial Findings

  First clues that Haemophilus may be present - 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 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

  
• Organism Requiring X Factor Only

  Aggregatibacter spp.
• Porphyrin Test


  Alternative method for differentiating the hemeproducing Haemophilus species, if they do not require X factor they are porphyrin positive (make X), δ-aminolevulinic acid (ALA) gets converted to porphyrins or protoporphyrins, Kovacs reagent produces red color in lower part of tube if porphobilinogen is present, Wood ultraviolet (UV) lamp causes porphyrin positive organisms to fluoresce reddish-orange
• CLSI guidelines for ID of commonly isolated bacteria - for respiratory tract or cerebrospinal fluid (CSF) specimens, gram-negative bacilli
• Organism Requiring X Factor Only