He, Who created the seven heavens in

layers. You will not find any flaw in the

creation of the All-Merciful. Look again—do
you see any gaps? Then look again and
again. Your sight will return to you dazzled
and exhausted!
(Qur'an, 67:3-4)
 Things to cover
o Definition of the Genus
o Habitat
o Culture Characteristics
o Infections Associated With Haemophilus
o Virulence factors of Haemophilus influenzae
o Pathogenesis and Immunity
o Clinical Manifestations, Hib
o Clinical Manifestations, Non-typable Strains
o Diagnosis
o Differential Characteristics of Medically Important Haemophilus
Species
o Biotyping
o Management
o Antimicrobial Susceptibility
o Prevention
 Definition of the Genus
 Small to medium sized coccobacilli or rods
 0.3-0.5 um x 0.5– 1.0 um with rounded ends
 Often markedly pleomorphic, some times filamentous
 Gram negative
 Non motile
 Nonsporeforming
 Nonacid-fast
 facultative anaerobe

 Difficult to stain by Gram stain

 Prolonged counterstaining for 5 – 15 min.with very
dilute carbol fuchsin often gives satisfactory
results
Definition cont…

Require one or both of two accessory factors-

Factor X (hemin)*
Factor V (NAD or NADP+)
5-10% CO2
Nitrate is reduced to nitrite
Oxidase and Catalase reaction varies among spp.; many
are positive
Obligate parasites of humans and animals, inhabiting
particularly mucous membranes
*See notes below
 Habitat
• Obligate parasites of the mucous membranes of humans and a
wide variety of animals
• Marked degree of host specificity
• Commonly inhabit the human upper respiratory tract and
mouth
• Rarely isolated from intestinal tract and vagina

Stains faintly with safranin counter-stain and it is easily

overlooked or misidentified in direct smear

• In BAP, RBCs are rich source of hematin but V factor is not

available unless blood is gently reheated after mixing with agar
(Chocolate Agar).
 Culture Characteristics
1) Growth factor requirements
• Will not grow in the absence of certain
factors present in blood--- ”blood loving”
(Haemophilus).
• Two accessory growth factors are required
• X factor:*
• Thermostable
• Plays a role as an enzyme in respiration.
• Porphyrin and hemin synthesis
*See notes below
 cont…
• V-factor:*
• Thermolabile
• Present in tissues of plants and animals
• Thought to be a vitamin ---So called as V-factor
• Synthesized by most bacterial spp. other than H
influenzae.
• S aureus … release V –factor
• Satellitsm
• e.g H influenzae and some strains of unrelated genera
like streptococci, neisseria and diphtheroids
•
*See notes below
 2) Other nutritional requirements

Vary among the spp.

H influenzae require Pantothenic acid,thiamin
and uracil.some strains also need purine and
cysteine
H ducreyi requires serum
H paragallinarum requires serum and NaCl
(though not halophilic).
 3) Physical requirements for growth

o Temp…35 – 37°C
o Minimum temp for growth is 20- 25°C
o H influenzae killed at 55 for 30min
o Grows better in aerobic than in anaerobic
o conditions
o Raised CO2 enhance growth of many spp.
 This is responsible for the phenomenon of “Satellitism” in which Hemophilus
influenzae forms colonies in the vicinity of Staphylococcal colonies.

BAP culture showing Haemophilus influenzae satelliting

around Staphylococcus aureus, Picture from CDC.
 S No Species Requires Hemolysis

X Factor V Factor

1 H influenzae
+ + -
2 H parainfluenzae
- + -
3 H ducreyi
+ - -
4 H hemolyticus
+ + +
5 H parahemolyticus
- + +
6 H aphrophilus
- - -
X = Hematin, V = NAD
 Virulence factors of Haemophilus Spp

• Capsule
• Fimbrial adhesin
• HMW1 and HMW2 adhesin
• Hap adhesin
• Hia and Hsf adhesins

• Opacity associated protein A (OapA)

– Mediate adherence to epithelial cells in culture
– Required for pharyngeal colonization in animal models
– Opaque colonial morphology on on trasparent growth medium

• Haemocin
– More than 90% of H influenzae type b
– Inhibits same or related spp.
– Not produced by encapsulated non type b or non typeable H influenzae
strains
Virulence factors cont…
• IgA protease

– Inactivates human IgA1 (accounts for over 90% of

IgA in oropharynx

• Lipooligosaccharide

– Differs from LPS by lacking the repeating terminal

side chain of the O or somatic antigen

• Outer membrane proteins

• 6-8 OMPs in the cell wall outer membrane

 Infections Associated With Haemophilus

• Meningitis
• Epiglottis
• Acute pharyngitis
• Pneumonia
 Pathogenesis
• H influenzae acts opportunistically as a secondary invader in a variety of
Respiratory Tract Infections usually preceded by viral infections.

• Infections most commonly associated with colonization are Meningitis,

Epiglottitis, Otitis media, Conjunctivitis, Pneumonia, Bacteremia, and
Arthritis.

• Colonization and Invasion

– H influenzae resides predominantly on the upper respiratory mucosal
surfaces
– Many strains are piliated
– Other molecules may also be involved
– Type b strains cause disease after invasion and hematogenous spread
to distant sites. Type b capsule is an important virulence factor
– Non-typeable strains cause disease by local invasion
 Pathogenesis
Localized Disease:
• Non-capsulated strains produce disease under circumstances in
which they get entrapped at a luminal site close to the upper
respiratory mucosa. It is usually associated with some
compromise of normal clearing mechanism due to a preceding viral
infection.

• This includes middle ear, sinuses or bronchi.

• Non-capsulated strains account for >90% of localized diseases.

• A family of non-pilus surface proteins (HMW-1, HMW-2) has

been identified. These proteins are not found in capsulated
strains.
 Immunity
• Antibody to capsular polysaccharide antigen( PRP) is important in
protection from infection. The age-related titer of this antibody
is inversely related to susceptibility to infection

• Maternal antibodies continue to decline after birth

• The child produces its own antibodies as a result of exposure to

H influenzae and the titer rises with age until (in the absence of
vaccination) protective levels reach at the age of 6 years after
which H influenzae infections are rare

• Non-capsular antigens have generated a considerable interest as

targets of immune response and as potential vaccine candidates.
Antibodies to OMP P1, P2 and P6 protect infant rats from
bacteremia by type b strains

• Secretory IgA in the naso-pharynx to P6 is associated with a

lower rate of colonization
 Clinical Manifestations, Hib
• Meningitis
– Most serious manifestation
– Primarily infection of infants younger than 2 years
– Clinically indistinguishable from other causes
– Sub-dural effusion is the most common complication. Suspicion is high
when no improvement in CNS signs after 2-3days of appropriate
antimicrobial treatment
– Mortality is 3-6%. Residual morbidity is up to 1/3 if carefully sought
for, most common being the sensori-neural hearing loss, partial
hearing loss and delays in language development

• Acute Epiglottitis
– A life-threatening infection involving cellulitis of the epiglottis.
Typically occurs in older children
– Can lead to upper airway obstruction if attempt to take a sample is
made.
– Sore throat and fever rapidly progress to dysphagia, drooling and
airway obstruction
 Clinical Manifestations, Hib
• Cellulitis
– Occurs in young children
– Face and/or neck area are commonly involved
– Bacteremia is usually present and 10% patients have additional
focus of infection elsewhere

• Pneumonia
– Also occurs in young children (<2years)
– Clinically indistinguishable from other bacterial pneumonias

• Less Common Infections

– Osteomyelitis, Septic Arthritis, Orbital Cellulitis, Endophthalmitis,
Urinary Tract Infection and Bacteremia without an identifiable
focus
– H aegyptius is associated with an acute purulent conjunctivitis.
– All these infections rare after 6 years of age. Also rare in
vaccinated children
 Clinical Manifestations, Non-Typable Strains
• Pneumonia
– Second most common cause of community acquired pneumonia
in adults especially with chronic obstructive airway disease
and AIDS.
– The underlying cause of bronchitis is related to chronic
damage resulting from smoking and other environmental
factors.
– Clinically not different from pneumonia of other etiologies,
but Gram stain may be suggestive and culture is diagnostic

• Otitis media
– One of the three most common causes of childhood OM
– Infants present with fever and irritability, older children
complain of pain.
– Viral URTI often precedes OM
– Tympanocentesis can be diagnostic but rarely done
 Clinical Manifestations, Non-typable Strains
• BRAZILIAN PURPURIC FEVER (BPF):
– H influenzae biovar aegyptius causes a fulminant pediatric
disease manifested by fever, hemorrhagic skin lesions,
septicemia, vascular collapse, hypotensive shock and death
usually within 48hours.

– It is preceded by purulent conjunctivitis which has resolved

before the onset of fever.

– Since its recognition in 1984, several outbreaks have

occurred in different cities of Brazil.

– Although invasive, these bacteria are not capsulated.

– Similar cases have been reported from Australia also but the
organism is genetically different from BPF strains.
 Diagnosis
• Gram stain of the clinical material may be suggestive.

• A positive culture is the gold standard for diagnosis

• The specimen needs to be inoculated on Chocolate Blood Agar

(CAP). Absence of growth on 5% Blood Agar and growth of
typical colonies on CAP is suggestive

• A Positive Oxidase Test, test for X and V factor dependence

and biochemical tests for H. influenzae confirm the diagnosis

• CSF
• Positive Gram stain for Gram Negative cocco-bacillary organisms
and subsequent culture of Gram Negative pleomorphic organism
is diagnostic.
• Gram stain is positive in ~80% of untreated cases
• Latex test for PRP is positive in ~95% cases
• PRP can be positive in other sterile body fluids
 Diagnosis
• With the exception of H ducreyi and H aphrophilus, all
Haemophilus spp require V factor and grow as satellite colonies
around Staphylococcal streak.

• Hemolytic Haemophilus spp do not show satellitism ?

 because the hemolysis secretes enough V factor to sustain
growth.

• H influenzae colonies are grayish, semi-opaque, smooth and

slightly convex. Diameter reaches 1-2mm in 24hours.

• Strains from meningitis and epiglottitis are almost always indole

positive (biovar II) and this gives them a smell similar to E coli.
Indole negative strains give a typical “mouse nest” smell.

• Colonies of H ducreyi are characteristically cohesive and can be

pushed intact across the surface of agar plate.
 Diagnosis
Microscopy of the Isolates:
• Gram stained smears show small
Gram negative cocco-bacilli with
various degree of polymorphism.

• X and V factor Requirement:

 Diagnosis
Porphyrin Test:
• The test is based on the observation that X factor independent strains
excrete porphobilinogen and porphyrin both of which are intermediates in
the hemin biosynthetic pathway when supplied with δ-aminolevulinic acid
(ALA).

• The substrate consists of 2mM ALA, and 0.8mM MgSO4 in phosphate

buffer at pH 6.9, distributed in 0.5ml quantities and stored at 4°C.

• A heavy suspension is made from agar plate and incubated for 4hours at
37°C. The mixture is exposed to UV light in a dark room and observed for
red fluorescence.

• A positive test shows presence of porphyrin, thereby depicting that growth

of bacteria is independent of factor X. Doubtful results may be re-
incubated for 24hours incubation.

• A positive test rules out H influenzae and H ducreyi.

 Diagnosis
• Several commercial kits are available for identification of
Haemophilus Spp once the V factor requirement has been
determined. API NH, Remel Haemophilus ID kit and Rapid ID NF
from Innovative Diagnostics etc.

• All automated systems are also equiped with panels identifying

Haemophilus Spp.

• A 16S rRNA based molecular test also identifies the Haemophilus if

facilities are available.

• Differentiation of H influenzae biotype III and H aegyptius may be

difficult by conventional methods. Poor in vitro growth, lack of
Xylose fermentation, and thin slender rod like morphology may help
in differentiation. OMP analysis are distinct for two species.
 Differential Characteristics of Medically Important Haemophilus Species
CO2
Porphyri X-Factor- V-Factor- Enhanceme Hemoly Catal
n Dependent Dependent nt sis[] ase
Haemophilus - + + + - +
influenzae

Haemophilus - + + - + +
haemolyticus

Haemophilus + - + - + V
parahaemolyticus

Haemophilus - + - V - -
ducreyi

Haemophilus + - + V - V
parainfluenzae

Haemophilus + - - + - -
aphrophilus

Haemophilus + - - + - -
paraprophilus
*
On horse and rabbit blood.
 Biotyping
• Three tests are used for biotyping of H influenzae
biotyping.
– Indole Test
– Urease Test
– Ornithine De-Carboxylase (ODC) Test

• The test media (0.3-0.5ml quantities) are inoculated with

loopful of growth and incubated for 4 hours. The test
for ODC may require additional incubation (up to 24
hours).
 Biotyping
DIFFERENTIATION OF BIOTYPES OF HAEMOPHILUS INFLUENZAE
BIOTYPE INDOLE UREASE ODC

BIOTYPE I + + +
BIOTYPE II + + -
BIOTYPE III* - + -
BIOTYPE IV - + +
BIOTYPE V + - +
BIOTYPE VI - - +
BIOTYPE VII + - -
BIOTYPE VIII - - -
*H. influenzae and H. aegyptius can be differentiated by analysis of OMP profile
 Haemophilus influenzae, Management
• Meningitis due to H. influenzae is treated with one of the newer
cephalosporins such as Ceftriaxone, Cefotaxime, or Cefuroxime.

• Alternative regimen is Ampicillin plus Chloramphenicol. Ampicillin

is discontinued if the isolate is resistant to it, or
Chloramphenicol is withdrawn if the isolate is Ampicillin
sensitive.

• Therapy is continued for up to two weeks

• Steroids Administration
– Studies have provided evidence that administration of steroids in meningitis
due to H. influenzae is beneficial with regard to reducing the frequency of
subsequent sequelae.

– Dexamethasone therapy is recommended for infants and children 2months

and older

– Mechanism is by reducing the inflammation induced by the mediators of

inflammation elaborated by the dying bacteria
 Antimicrobial Susceptibility
• CLSI recommends testing
• Ampicillin, chloramphenicol, a third-generation cephalosporin,
and meropenem
• Resistance to ampicillin may be as high as 40-60% in the United
States,.
• Resistance to ampicillin is usually mediated by
– the production of β-lactamase;
– alterations in outer membrane permeability or
– affinity to penicillin-binding proteins.

• Susceptibility testing of H. influenzae requires the use of

Haemophilus test medium (HTM).

• The recommended treatment for H. ducreyi infection is

erythromycin; alternative agents include azithromycin,
ciprofloxacin, ceftriaxone, amoxicillin–clavulanate, and
trimethoprim–sulfamethoxazole.
 Haemophilus influenzae, Management
• Invasive infections other than meningitis are treated
with the same antimicrobial agents.

• Epiglottitis is a medical emergency and maintenance of an

airway is critical.

• Duration of therapy is determined by the clinical course.

1-2 weeks treatment is usually enough.

• Many infections caused by non-typeable strains can be

treated with oral antimicrobials.

• About 25% strains are beta lactamase producer and such

cases can be treated with a variety of agents including,
trimethoprim-sulfamethoxazole, amoxy-clav,
erythromycin, fluoroquinolones, and newer macrolides
 Haemophilus influenzae, Prevention
• Chemoprophylaxis
– Risk of secondary infection is greater in household contacts
– All member of the household with a susceptible child should receive
Rifampin
– No prophylaxis is required if the children <4 year age are
vaccinated
– Index case also should receive Rifampin for eradication of the
organism from naso-pharynx

• Earlier Polysaccharide (PRP) vaccine had no benefit in children

younger than 18months

• Conjugate vaccines prevent infections by H. influenzae type b in

infants and children

• Three dose series can be started at 2 months age. If the first

dose is given after 7 months age, two doses are given and after
12 months only one dose. All receive a booster at 15 months of
age or later
 Haemophilus ducreyi
• It causes the STD “Soft Chancre” or “Chancroid”.

• The genital lesion starts as tender papule that becomes pustule

and then ulcerates over a course of 2 days.

• Lesions may merge to form large ulcers with involvement of

inguinal lymph nodes.

• It is a major cause of genital ulcers in Asia, Africa and South

America.

• The disease has earned an increased interest because of its

association with HIV transmission.

• Extra genital lesions may occur but are extremely rare.

 Haemophilus ducreyi
• The specimen should be taken from base and
undermined edge of the lesion with a saline or
broth-moistened swab.

• Cultivation may be supplemented by aspiration

from an involved LN.

• Direct plating on selective media is advisable as

it results in acceptable rates of recovery.
• Specimen must be refrigerated if transport
time is longer than a few hours.
Large fluctuant buboes should be
drained with local anesthesia and a
large-gauge needle inserted
through surrounding healthy skin

• There is no immunity following the infection.

• Treatment with Ceftriaxone, oral SXT or

Macrolides often results in healing in two weeks.
 Other Haemophilus Spp
Haemophilus aphrophilus:
• This organism is sometimes encountered in infective
endocarditis and pneumonia.

• It is part of normal oral and URT Tract flora.

Haemophilus hemolyticus:
• It is the most markedly hemolytic of Haemophilus spp.

• It occurs as normal flora of respiratory tract and occasionally

in association with URT infections of moderate severity in
children.

Haemophilus parainfluenzae:
• It is part of normal flora and resemble H influenzae. It
requires V factor only.

• It is rarely encountered in infective endocarditis.

"The man who says it cannot
be done should not interrupt
the man doing it."

Chinese Proverb