Genus Haemophilus

General characters
 Gram negative coccobacilli and/or bacilli,
facultatively anaerobic, non-mobile, non-
sporogenous
 Virulent strains are capsulated
 Fastidious – need growth factors from blood
in order to cultivate:
 X Factor = haemin or other iron containing
porphyrins
 V Factor = NAD
Morphology
Morpholog

Microscopy:
 Polymorphism pending on growth
conditions and age of culture
 Species specific aspects
 Borderline Gram negative:
Decolorate harder
Counterstain harder: better to use fucsin, NOT
SAFRANIN
Risk factors and types of infections
Risk factors:
 Extreme ages
 Immune system deficiency

Types of infections:
 Respiratory tract and neighbouring sites pneumonia, meningitis,
acute epiglottitis, otitis media: mostly incapsulated strains
 Cellulitis
 Bacteriemia, septicemia
 Arthritis, pericarditis, endocarditis, osteomyelitis
 Infections of genitals, infections in pregnant women, women in
childbed and fetus
 Infections of wounds
 Digestive and billiary infections
Taxonomy

 Pasteurellaceae Family
Genus Haemophilus
Genus Actinobacillus
Genus Pasteurella
 Relatives:
Enterobacteriaceae
Vibrionaceae
Aeromonaceae
Habitat

 Humans, mammals, birds, fish, reptiles:

Normal microbiota of respiratory tract, genitals,

intestine, dental plaque

Exception:
H.aegyptius and H. ducreyi: always pathogens
Species pathogenic to humans
 H. influenzae - especially serovar b
 Haemophilus = “blood lover”
 Influenzae = “of influenza”
 Great flu epidemic from 1889 – 1892 – Pfeiffer described H. influenzae
in patients lungs
 Smith, Andrewes an Laidlaw – established the secondary role of H.
influenzae (bacterial complication of influenza)
 H. parainfluenzae
 H. aphrophilus
 H. ducreyi (chancroid, soft chancre or ulcus molle);
 H. aegyptius -- bacterial (purulent) conjunctivitis and
Brasilian purpuric fever
Note: Species dependent only on Factor V – “para”
(exception H. segnis)
H. influenzae –
first microorganism
whole genome sequenced, 1995
Growth
 Enriched media, supplemented with peptones,
glucose etc.
 Need X Factor and V Factor; sometime only V Factor
 Factor X, termostable (45 min to 120 o C) = hemin

 Factor V, termolabile (15 min to 90 o C) = NAD or NADP –

inactivated by pyrophosphatase and nicotin amin deoxi
ribonuclease
Alternatives to provide X and V factors:
 Heating of blood agar to obtain chocolate blood agar
 Supplements: 5% blood, factor X and V, Fildes or
Levinthal supplements
 Staphylococcus streak or satellite test
Staph streak technique
or satellite test
Cultivability peculiarities
 Capnophilic species: H. ducreyi,
aphrophylus, certain H. parainfluenzae
 Horse or rabbit blood
 Need humidity
 Prolonged incubation : 48-72 h after
antibiotic treatment and 5 days for H.
ducreyi
 Selective media for highly contaminated
samples
Pathogenicity factors
 Capsule
 Endotoxin differs of enterobacteria endotoxin:
lipooligosaccharide
instead of LPS in external membrane – role in
inflammation
 Outer Membrane Proteins (OMPs) P1 and P2
 All virulent strains produce a neuraminidase and an
IgA protease
 Fimbria increase adherence to mucosal surfaces
 There is a receptor for fimbria on erithrocytes:
antigen Anton
Pathogenesis
 Adherence

 Reaches submucosal layer – lymph and blood

vessels

 Blood stream (resistance by capsule)

 Reaches CSF (hematoencephalic barrier)

 Endotoxin: fever, Intra Vascular Disseminated

Coagulation
Clinical and laboratory diagnostic
 Clinical: history, physical signs,
symptomatology
 Positive blood cultures in over 50% of
symptomatic cases, except cases with
conjunctivitis
 Presence of Polyribitol phosphate in
serum and CSF, which concentrate in
urine in more than 95% of H. Influenzae
meningitis cases
Sampling

 Samples:
sputum,
CSF,
blood,
otic secretion,
pus from cellulitis or wounds, vaginal or
urethral secretions etc.
Microscopy

Haemophilus influenzae microscopy

Media for isolation

 Haemophilus media:
Blood agar with horse or rabbit blood
Chocolate agar
 XV agar
 XV differential agar
Levinthal agar
Fildes agar
Selective media
Special requirements Haemophilus
influenzae

Haemophilus influenzae requires X

and V factors for growth.
In this culture Haemophilus has
only grown around the paper disc
that has been impregnated with X
and V factors. There is no
bacterial growth around the discs
that only contain either X or V
factor.
Satellitism Staphylococcus aureus
 H. influenzae will grow in the
hemolytic zone of
Staphylococcus aureus on
blood agar plates; the
hemolysis of cells by S. aureus
releases factor V which is
needed for its growth. H.
influenzae will not grow
outside the hemolytic zone of
S. aureus because of the lack
of nutrients such as factor V in
these areas
Identification and typing
 Preliminary identification: comparative evaluation of
growth on media with V, X, VX factors in aerobic and
CO2 atmosphere; satellitism
 Final identification: stepwise, using biochemical tests:
amino acids, saccharides; 8 biotypes of H. influenzae,
pending on indol, urease, ornitin reactions
 AST special media
 Typing:
 Antigenic: based on capsular, OMP (Outer Membrane
Proteins), Lipooligosaccharides (LOSs) and/or fimbrial
proteins
 DNA - ribotyping
Epidemiology and prevention

 Disease incidence is inversely

proportional with blood bactericidal
antibodies titre, acquired passively
from mother or actively synthesized
 If no immunisation, Ab titer in children
between 2 months and 3-4 years of
age is minimal there is need to
vaccinate at 2 months