Haemophilus

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Haemophilus
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Introduction

• During the influenza pandemic of 1918 a
  pleomorphic Gram negative bacterium was
  isolated from the respiratory tract of most of
  the patients who were dying of influenza
• The bacterium grew only on media containing blood
  from rabbits, horses, and cows. It did not on
  media containing unheated sheeps blood unless it
  was first heated to 80oC, a temp that releases X
  V factors without destroying them. This was
  the 1st use of what we now call chocolate blood
  agar (CBA).
• The bacterium was named Haemophilus, or blood
  loving because it grew only on media containing
  blood. The species name influenzae was used
  because it was mistakenly presumed to cause
  influenza.
• In the 1930s, after cell cultures and the
  electron microscope were invented, it was
  demonstrated that influenza is caused by a virus,
  the influenza virus (orthomyxovirus).

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• Blood of all animal species contains 2 growth
  factors, both of which are stable at 80oC.
• The labile factor is extracellular but is
  destroyed by a heat labile enzyme found in
  sheeps blood, but not in rabbit, horse, or cow
  blood. This is NAD and is known now as V factor.
• 2) The stable factor is stable in the presence
  of the enzyme found in sheeps blood, but it is
  intracellular, and is released from RBCs heated
  at 80oC. This is now known to be heme, and is
  referred to as X factor.
• H. ducreyi requires the addition of X factor but
  not V factor. H. aphrophilus is unable to
  synthesize X factor on primary cultures but gains
  the ability upon subculture. Species names with
  the prefix para cannot synthesize V factor but
  can synthesize X factor. All other Haemophilus
  species and a few other species require both
  growth factors because they lack the enzymes to
  synthesize them.

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Introduction

• The inactivating enzyme in sheep's blood but not
  the blood of rabbits, horses, or cows is NADase
• H. influenzae will grow on unheated SBA if NAD
  is added in sufficient quantities to overcome
  NADase. Alternatives to adding NAD are to add
  the B vitamin (not really) niacin, or by growing
  an NAD-producing microbe along with H.
  influenzae. Various yeast species are the best
  microbes to use for this purpose.
• To test an organism for the necessity of added X
  /or V factors, conduct a cross streak of the
  organism with S. aureus on agar containing blood
  previously heated to 100oC. The isolate will
  grow as tiny satellite colonies next to the S.
  aureus
• Even though the staph streak method will allow
  H. influenzae and other species to grow, the
  colonies remain tiny.
• H. influenzae grows much larger colonies on
  chocolate agar

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Cross streak technique for growth factors
Satellite colonies
Staphylococcus Growth
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Introduction

• The modern version of chocolate agar, which
  allows H. influenzae to grow up to 3mm diameter
  size colonies in 48 hours, involves adding
  hemoglobin powder to a basal agar medium such as
  TSA. Autoclave it, cool to 50oC, and add
  filter-sterilized yeast extract as an NAD source
  to the molten 50oC agar base.
• As said earlier, H. influenza does not cause
  influenza, but can cause secondary bacterial
  infections including pneumonia and septicemia.
• If the microbiologists who misnamed the bacterium
  had followed Kochs postulates, another species
  name almost certainly would have been chosen.
• As we will discuss later, the primary pathology
  associated with the b strain of this organism
  is meningitis. This strain is now referred to as
  HIb.

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General characteristics

• The genus Haemophilus consists of Gram negative,
  pleomorphic rods. In clinical specimens they
  range from small coccobacilli to long filamentous
  rods, and are usually encapsulated.
• They are nonmotile and aerobic to facultatively
  anaerobic
• Most species are catalase and oxidase positive
  (may be weak)
• There are 10 species of Haemophilus associated
  with humans
• Most, including H. influenzae, are part of the
  normal microbiota of the URT and other mucous
  membranes.
• The two major pathogens are H. influenzae and H.
  ducreyi. The others are occasionally associated
  with opportunistic infections
• Several Haemophilus species are hemolytic on
  rabbit or horse blood

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Clinical specimens and culture

• Haemophilus species can be isolated from various
  clinical specimens including blood, CSF,
  middle-ear exudate, joint fluids, URT and LRT,
  conjunctivae, vagina, and wounds and abscesses
  (although not routinely plated unless
  specifically requested)
• H. ducreyi is more fastidious than other
  Haemophilus species. If the physician suspects H.
  ducreyi, the media should be incubated for at
  least one week
• Media for isolating Haemophilus should be
  incubated in a humid environment rich in carbon
  dioxide
• Colonies of Haemophilus are 1-3 mm in diameter in
  24-48 hours of incubation. On CBA they are
  slightly convex moist, circular smooth and
  translucent, and have a distinct mousy or a
  very faint bleach-like odor
• Highly virulent encapsulated strains of H.
  influenzae produce a slightly grayish and very
  moist appearing colonies

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Identification

• The first clue that Haemophilus may be present in
  pure culture is good growth on CBA and no
  growth on SBA. If the culture is from a
  non-sterile site (mixed culture), tiny satellite
  colonies growing around colonies of other species
  is another clue
• The most virulent H. influenzae strains or
  biotypes can be detected using the spot indole
  test and oxidase test. Most virulent strains of
  H. influenzae are positive for both tests
• Most virulent strains of H. influenzae also
  produce a very strong urease. Actual growth is
  not necessary if a heavy inoculum is used - the
  transferred urease will give a rapid positive
  reaction
• The next step is to confirm X, V, or XV
  requirements. The traditionally test involves
  placing paper discs (or strips) containing X and
  V on media devoid of both factors. Discs of both
  factors are arranged on the agar close together
  and distant from each other.

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Identification

• Organisms that grow only between the closely
  placed disks require both factors.
• Those that grow only around the V disks cannot
  synthesize V (require V)
• Those that grow only around the X disks cannot
  synthesize X (require X)
• When identifying Haemophilus species using X and
  V discs a very light inoculum should be used
  (less than 0.5 MacFarland)
• When picking the colony be careful not to touch
  the medium because carry-over of factors from
  the media can result in erroneous results

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X and V Requirements
Growth Between X and V Disk Only
X
V
X
V
Requires X and V
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X and V Requirements
Growth Around X Disks only
V
X
X
V
Requires X only
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X and V Requirements
Growth Around V Disks only
X
V
X
V
Requires V only
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Identification

• Most clinical microbiology labs now use some
  commercial system to identify Haemophilus species
  such as the quad plate
• One quadrant of a divided plate contains only X,
  one contains only V, one contains X and V, and
  one contains rabbit or horse blood for detection
  hemolysis. The plate is read very much like the
  paper strip method previously described.
• Many microbiologists abandoned the use of strips
  and media containing X and V because they claim
  that it is impossible to avoid carry-over,
  especially for the X factor
• Instead, they determine if an isolate can
  synthesize the X factor by using the porphyrin
  test (recall that heme is derived form a
  protoporphyrin)

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Identification

• The porphyrin tube test is performed by making a
  heavy suspension in buffered aminolevulinic acid
  (ALA) solution
• If the isolate can synthesize heme, it will
  enzymatically convert ALA to porphobilinogen
  (PBG) - the lack of the enzymes to do this limits
  all Haemophilus that cannot synthesize heme.
  From PBG an organism can produce protoporphyrin
  and then heme
• If PBG is produced, a red color will develop in
  the presence of Ehrlichs reagent (pDAB) recall
  from urinalysis
• Protoporphyrin can also be detected rather easily
  because it fluoresces red when exposed to
  ultraviolet light

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Porphyrin Test
Bacterial enzymes
Bacterial enzymes
ALA PBG Protoporphyrin
Red Fluorescence (UV light)
Red Color (pDAB)
Interpretation Organisms that produce a red
color with either pDAB or UV light can
synthesize heme heme does not have to be add to
a medium to grow this organism it does not
require X. Organisms failing to produce the red
color(s) do require X to be added to a growth
medium
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Porphyrin Tube Test
ALA medium exposed to pDAB or UV light
After addition of pDAB If red appears, it does
not require X If no red appears it does
requireX
UV Light If red appears, it does not require
X If no red appears it does require X
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Identification

• The UV test can be performed on an agar plate
  containing ALA
• The unknown is band streaked on the plate
  after overnight incubation the surface of the
  plate is exposed to UV light
• A red fluorescence indicates the organism can
  synthesize porphyrin (does not require X)
• No red fluorescence indicates the organism cannot
  synthesize porphyrin (does require X)
• Several commercial ID products use miniaturized
  tubes with dehydrated substrates. RapID NH is
  one such product
• It can identify Haemophilus and other fastidious
  coccobacilli as well as Neisseria and related
  organisms

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Plate Porphyrin Test
Bacteria band streaked across a plate
containing ?- ALA
Unknown A
Unknown B
A Red Fluorescence with UV light (Does not
require X) B No red fluorescence with UV
light (does require X)
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Identification of Haemophilus
Do require X (can not synthesize their own
porphyrin) and do require V (can not
synthesize their own NAD)
Hemolysis1 Indole Ornithine Urea
H. influenzae Biotype I -
H. influenzae
Biotype II - -
H. influenzae Biotype III2 - -
- H. hemolyticus
/- -
1Horse or rabbit blood 2Some strains of Biotype
III were formerly called H. aegyptius, a major
causes of pinkeye
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Identification of Haemophilus
Do not require X (can synthesize their own
porphyrin) Do require V (can not synthesize
their own NAD)
Hemolysis1 Indole Ornithine Urea
H. parainfluenzae -
- H.
parahaemolyticus -
/- H. segnis -
- - - H.
paraphrophilus - -
-
1Horse or rabbit blood
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Identification of Haemophilus
Does require X (can not synthesize its own
porphyrin) Does not require V (can synthesize
its own NAD)
Hemolysis1 Indole Ornithine Urea
H. ducreyi /- -
- -
Does not require X1 (can synthesize its own
porphyrin does not require V ( can
synthesize its own NAD)
Hemolysis2 Indole Ornithine Urea
H. aphrophilus - -
- -
1 May require heme on primary isolation
2Horse or rabbit blood
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Virulence factors of H. influenzae

• The following virulence factors play a role in
  the invasiveness of this organism and the
  initiation of infection
• Type b polysaccharide capsule attachment
  anti-phagocytosis appears critical for
  virulence
• Endotoxin (LPS) plays role but not understood
• IgA protease neuraminidase (like
  orthomyxovirus) produced by all virulent strains
  but role in virulence unknown
• Adherence factors fimbriae and protein
  adhesins required for initial colonization

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Infections caused by H. influenzae
Encapsulated type b (pediatric)
Nonencapsulated (pediatric)
Nonencapsulated (adults)
Meningitis Septicemia Cellulitis Epiglottitis
Conjunctivitis
Otitis media
Pneumonia Bronchitis Sinusitis
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Infections caused by other Haemophilus

• H. aegyptius, a subspecies of H. influenzae
  (biotype III), causes contagious purulent
  conjunctivitis (also known as pinkeye)
• H. ducreyi causes chancroid, a highly
  communicable sexually transmitted disease that is
  most prevalent in Africa Asia, but is probably
  under-reported in the US due to misidentification
  
• H. aphrophilus, H. parainfluenzae, and H.
  paraphrophilus cause endocarditis (rarely)

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Antimicrobial therapy

• Ampicillin has historically been the drug of
  choice for serious invasive H. influenzae
  infections, especially meningitis
• Since the mid-1980s 10 to 20 of Haemophilus
  species have become ampicillin resistant (plasmid
  mediated beta lactamase)
• Chloramphenicol was used as the back-up
  antibiotic for ampicillin resistant strains
• Chloramphenicol resistant strains have now begun
  to appear
• Third generation cephalosporins are now the drugs
  of choice
• Also Trimethoprim-sulfamethoxazole or
  ciprofloxicin are clinically effective for
  strains that are ampicillin and chloramphenicol
  resistant

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Antimicrobial testing

• All isolates from blood or CSF, and isolates
  involved in life threatening infections are
  tested for beta lactamase
• The most widely used test employs a chromogenic
  cephalospirin substrate, Cefinase
• If the isolate does not produce beta lactamase
  (negative test result) ampicillin should be
  effective
• If the beta lactamase test is positive a modified
  Kirby Bauer disk diffusion test is performed
  utilizing a Haemophilus test medium (HTM) that
  contains X V rather than the regular Kirby
  Bauer medium, Mueller-Hinton.
• The Kirby Bauer test determines extent of
  resistance ie. MIC of antimicrobial agent, in
  this case ampicillin

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Beta Lactamase Testing
Beta lactamase
Chromogenic Hydrolysis of
Colored Cephalosporin
Beta Lactam Ring products
Reagent Cefinase

released



If beta lactamase positive a red color will
develop within 30 seconds
Remove growth from plate
Rub on Cefinase disk
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Antimicrobial testing

• The E-test on HTM agar can also be used to test
  for antimicrobial susceptibility of Haemophilus
• The E-test uses a plastic strip that is
  impregnated with a concentration gradient of
  antimicrobial agents
• The strip is placed on a plate of HTM agar that
  has been spread-plate inoculated with a 0.5
  MacFarland broth culture
• The antimicrobial gradient diffuses into the agar
  
• Susceptible strains forms an elliptical pattern
  of inhibition
• The point near the low concentration end of the
  strip that is intersected by the zone of
  inhibition represents the minimal inhibitory
  concentration (MIC) of the antimicrobial agent

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E-Test
E
High concentration end of the strip
Zone of Inhibition
O.50ug/ml line
Bacterial Growth
Low concentration end of the strip