Haemophilus and the HACEK Organisms

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Haemophilus and the HACEK Organisms

• Dr. John R. Warren
• Department of Pathology
• Northwestern University
• Feinberg School of Medicine
• June 2007

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Characteristics of Haemophilus

• Small, pleomorphic gram-negative coccobacilli
• Positive for cytochrome oxidase
• No growth on MacConkey agar
• Growth in culture requires exogenous hemin
  (oxidized ferroprotoporphyrin) (X factor) and/or
  nicotinamide adenine dinucleotide (NAD) (V
  factor)

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Characteristics of Haemophilus

• Sheep blood contains NADase, a V
  factor-destroying enzyme, and consequently
  clinical strains of Haemophilus do not grow on
  standard 5 sheep blood agar
• Heating sheep blood agar at 80oC denatures NADase
  and heat-lysis of red cells releases free NAD
  (chocolate agar)

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Characteristics of Haemophilus

• Growth of Haemophilus on chocolate agar in the
  presence of 5-10 CO2 (capnophilia)
• Contemporary chocolate agar is a synthetic mix
  of NAD, hemoglobin, vitamins (cobalamin, thiamine
  hydrochloride), minerals (iron, magnesium),
  cysteine, glutamine, and glucose

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Most Common Species of Haemophilus

• Haemophilus influenzae
• Haemophilus parainfluenzae
• Haemophilus aphrophilus
• Haemophilus ducreyi

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Haemophilus Natural Habitats

• Normal inhabitant of the upper respiratory,
  gastrointestinal, and genital tracts of humans
  except Haemophilus ducreyi
• Haemophilus ducreyi found only in humans during
  disease (not normal microbial flora)

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Haemophilus Modes of Infection

• Encapsulated strains of Haemophilus influenzae
  associated with invasive infection caused by
  person-to-person spread of H. influenzae due to
  inhalation of infectious respiratory droplets
• Type b H. influenzae was most commonly associated
  with disease prior to conjugate vaccine but
  prevalence has declined with advent of vaccination

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Haemophilus Modes of Infection

• Prevalence of invasive type b H. influenzae
• disease (meningitis, epiglottitis, pneumonia,
• septic arthritis, osteomyelitits, facial
• cellulitis) in children less than 5 years of
• age decreased 95 in the United States
• from 1987 to 1993 (41 cases per 100,000 to
• 2 cases per 100,000) due to childhood
• vaccination

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Haemophilus Modes of Infection

• Endogenous non-typeable strains of Haemophilus
  influenzae associated with invasive infection in
  debilitated adults
• Haemophilus ducreyi sexually transmitted

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Haemophilus Types of Infectious Disease

• Encapsulated (types a-f) strains of H. influenzae
  produce invasive infection (pneumonia,
  meningitis, epiglottitis, and bacteremia)
• Unencapsulated (non-typeable) strains of H.
  influenzae cause otitis media in children, and
  lower respiratory tract infections (acute
  tracheobronchitis, pneumonia) in children and
  adults

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Haemophilus Types of Infectious Disease

• Haemophilus influenzae biogroup aegyptius
• (formerly H. aegyptius) lacks a capsule but
• capable of invasive infection, including
• Purulent conjunctivitis
• Sepsis syndrome in children (1-4 years of age)
  following purulent conjunctivitis with mortality
  as high as 70 (Brazil and Australia)

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Haemophilus Types of Infectious Disease

• Respiratory infection by non-typeable H.
  influenzae associated with underlying conditions
  (cystic fibrosis, chronic obstructive pulmonary
  disease, malignancy)
• H. parainfluenzae of low virulence with sporadic
  cases of endocarditis and bacteremia

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Haemophilus Types of Infectious Disease

• Haemophilus aphrophilus an uncommon cause of
  slowly progressive (subacute) endocaridits and
  brain abscess (member of the HACEK group)
• Haemophilus ducreyi causes chancroid, a sexually
  transmitted disease (400 cases annually in the
  US) with shallow and painful genital ulcers
  associated with inguinal lymphadenitis (bubo
  formation)

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Haemophilus Types of Infectious Disease

• Haemophilus haemolyticus, H.
• parahaemolyticus, H. segnis,
• and H. paraphrophilus rarely
• produce human infection.

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Haemophilus Species Identification

• Preliminary findings small faintly-staining
  (with safranin) gram-negative coccobacillary to
  filamentous rods that grow on chocolate agar but
  not sheep blood agar (except H. aphrophilus that
  with passage in culture grows on both)
• X and V factor requirements determined using X,
  V, and XV factor impregnated paper strips on
  Mueller-Hinton agar

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Haemophilus Species Identification

• Growth requirement for X and V factors, no ?
  hemolysis on horse blood agar ? Haemophilus
  influenzae
• Growth requirement for V factor, no ? hemolysis
  on horse blood agar ?Haemophilus parainfluenzae

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Haemophilus Species Identification

• Growth requirement for X and V factors, ?
  hemolysis on horse blood agar ? Haemophilus
  haemolyticus
• Growth requirement for V factor, ? hemolysis on
  horse blood agar ?Haemophilus parahaemolyticus

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Haemophilus Species Identification

• Growth requirement for X factor ? Haemophilus
  aphrophilus (requirement lost upon
  subcultivation)

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Haemophilus Species Identification Using the
Quad Plate (Remel)1

• Quad 1-Horse Blood Agar
• Quad 2-X Factor 1ßß-hemolysis, Noßno
• Quad 3-V Factor ß-hemolysis, ggrowth,
• Quad 4-XV Factor ngno growth
• 1 2 3 4
• H. influenzae Noß ng ng g
• H. haemolyticus ß ng ng g
• H. parainfluenzae Noß ng g g
• H. parahaemolyticus ß ng g g

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Haemophilus Species Identification Using
Biochemicals1

• X V Hem Glu2 Suc2 Lac2 Man2
• H. inf
  
• H. para
  
• H. haem
  
• H. aph3
  
• 1XX factor, VV factor, Hemß-hemolysis on horse
  blood agar
• 2Glucose (Glu), sucrose (Suc), lactose (Lac), and
  mannose (Man) broth supplemented with X and V
  factors
• 3X factor requirement often lost upon
  subcultivation

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Haemophilus influenzae Biotypes

• Biotypes II and III associated with respiratory
  tract, middle ear, and eye infections
• Biotype III includes Haemophilus influenzae
  biogroup aegyptius (Koch-Weeks bacillus)
• Biotype IV associated with genital infections,
  neonatal sepsis (mortality 50-90) and postpartum
  sepsis associated with endometritis
• Most serotypes a, b, and f strains biotype I
• Serotype c strains usually biotype II
• Serotype d and e are biotype IV

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Haemophilus influenzae Biotypes

• Indole Urease Ornithine
• I
• II
• III
• IV
• V
• VI
• VII
• VIII

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HACEK Organisms

• HHaemophilus aphrophilus
• AActinobacillus actinomycetemcomitans
• CCardiobacterium hominis
• EEikenella corrodens
• KKingella kingae

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HACEK Organisms

• Slow growing, capnophilic, gram-negative bacteria
  that cause endocarditis
• Clinical suspicion of endocarditis with negative
  blood cultures after standard 5 days of
  incubation (incubate blood cultures for 2 weeks)

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HACEK Organisms

• H A1 C2 E3 K4
• Mac /5 /
• Oxidase /
• Catalase
• Indole
• Nitrate
• Glucose
• Mannitol /
• Sucrose
• 1Colonies show central opaque dot that with
  incubation forms a star-like configuration like
  crossed cigars visible on clear agar medium
  such as brain heart infusion (supplemented with
  serum) at 100X magnification may show light
  growth on MacConkey agar
• 2Irregularly-staining gram-negative rods with
  bulbous (swollen) ends indole detected by xylene
  extraction
• 3Cultures smell of hypochlorite (bleach)
• 4Colonies show small but distinct zones of
  ß-hemolysis
• 5X-factor requirement lost with passage in culture

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Recommended Reading

• Winn, W., Jr., Allen, S., Janda, W., Koneman,
• E., Procop, G., Schreckenberger, P., Woods,
• G.
• Konemans Color Atlas and Textbook of
• Diagnostic Microbiology, Sixth Edition,
• Lippincott Williams Wilkins, 2006
• Chapter 9. Miscellaneous Fastidious
  Gram-Negative
• Bacilli

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Recommended Reading

• Murray, P., Baron, E., Jorgensen, J., Landry,
• M., Pfaller, M.
• Manual of Clinical Microbiology, 9th
• Edition, ASM Press, 2007
• Kilian, M. Chapter 41. Haemophilus.
• von Graevenitz, A., Zbinden, R., and Mutters, R.
  Chapter 40. Actinobacillus, Capnocytophaga,
  Eikenella, Kingella, Pasteurella, and Other
  Fastidious or Rarely Encountered Gram-Negative
  Rods