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Bacteriology 330 Lecture Topics: Haemophilus influenzae
©1999 Kenneth Todar University of Wisconsin Department of Bacteriology
Introduction
Haemophilus influenzae is a small, nonmotile Gram-negative
bacterium in the family Pasteurellaceae, on the level with the Vibrionaceae
and the Enterobacteriaceae. The family also includes Pasteurella
and Actinobacillus, two other genera of bacteria that are parasites of
animals. Encapsulated strains of Haemophilus influenzae isolated
from cerebrospinal fluid are coccobacilli, 0.2 to 0.3 to 0.5 to 0.8 um, similar
in morphology to Bordetella pertussis, the agent of whooping cough. Non
encapsulated organisms from sputum are pleomorphic and often exhibit long
threads and filaments. The organism may appear Gram-positive unless the Gram
stain procedure is very carefully carried out. Furthermore, elongated forms
from sputum may exhibit bipolar staining, leading to an erroneous diagnosis of Streptococcus
pneumoniae. .
Figure 1. Gram stain of Haemophilus influenzaefrom sputum
H. influenzae is highly adapted to its human host. It is present in
the nasopharynx of approximately 75 percent of healthy children and adults. It
is rarely encountered in the oral cavity and it has not been detected in any
other animal species. It is usually the non encapsulated strains that are
harbored as normal flora, but a minority of healthy individuals (3-7 percent)
intermittently harbor H. influenzae type b (Hib) encapsulated strains in
the upper respiratory tract. Pharyngeal carriage of Hib is important in the
transmission of the bacterium. The success of current vaccination programs
against Hib is due in part to the effect of vaccination on decreasing carriage
of the organism.
What's in a name?
Haemophilus influenzae is widespread in its distribution among the
human population. It was first isolated by Pfeiffer during the influenza
pandemic of 1890. It was mistakenly thought to be the cause of the disease
influenza, and it was named accordingly. Probably, H. influenzae was an
important secondary invader to the influenza virus in the 1890 pandemic, as it
has been during many subsequent influenza epidemics. In pigs, a synergistic
association between swine influenza virus and Haemophilus. suis is
necessary for swine influenza. Similar situations between human influenza virus
and H. influenzae have been observed in chick embryos and infant rats.
Haemophilus "loves heme", more specifically it requires a
precursor of heme in order to grow. Nutritionally, H.aemophilus influenzae
prefers a complex medium and requires preformed growth factors that are present
in blood, specifically X factor (i.e., hemin) and V factor (NAD
or NADP). In the laboratory it is usually grown on chocolate blood agar which
is prepared by adding blood to an agar base at 80 degrees. The heat releases X
and V factors from the RBCs and turns the medium a chocolate brown color. The
bacterium grows best at 35-37 degrees and has an optimal pH of 7.6. Haemophilus
influenzaeis generally grown in the laboratory under aerobic conditions or
under slight CO2 tension (5% CO2), although it is capable
of glycolytic growth and of respiratory growth using nitrate as a final
electron acceptor.
In 1995, Haemophilus influenzae was the first free-living organism to
have its entire chromosome sequenced, sneaking in just ahead of Escherichia
coliin that race, mainly because its genome is smaller in size than E.
coli's. For a relatively obscure bacterium, there was already a good
understanding of its genetic processes, especially transformation.
Figure 2. A map of the circular chromosome of Haemophilus influenzae illustrating
the location of known genes and predicted coding regions
Observations of genetic transformation in Haemophilus have included
drug resistance and synthesis of specific capsular antigens. The latter is
thought to be the main determinant of H. influenzae.
Transformation in Haemophilus influenzaeoccurs by several different
mechanisms and is more efficient than in enteric bacteria. When developing
competence, the bacterium develops membranous "blebs" in the outer
membrane that contain a specific DNA-binding protein. This outer membrane
protein recognizes a specific 11-base pair sequence of DNA nucleotides that
appears in Haemophilus DNA with much higher frequency than in other
genera of bacteria. There is some evidence that Haemophilus is able to
undergo both interspecies and intraspecies transformation in vivo (in host
tissues). The restriction endonucleases from Haemophilus, e.g. Hind
III, are widely used in biotechnology and in the analysis and cloning of
DNA.
Pathogenesis
The pathogenesis of H. influenzae infections is not completely
understood, although the presence of the type b polysaccharide capsule
is known to be the major factor in virulence. Encapsulated organisms can
penetrate the epithelium of the nasopharynx and invade the blood capillaries
directly. Their capsule allows them to resist phagocytosis and
complement-mediated lysis in the the nonimmune host. Nontypable (non
encapsulated) strains are less invasive, but they are apparently able to induce
an inflammatory response that causes disease. Outbreaks of H. influenzae type
b infection may occur in nurseries and child care centers, and prophylactic
administration of antibiotics been warranted. Vaccination with type b
polysaccharide ( in the form of Hib conjgate vaccines) is effective in
preventing infection, and several vaccines are now available for routine use.
Naturally-acquired disease caused by H. influenzae seems to occur in
humans only. In infants and young children (under 5 years of age), H. influenzae
type b causes bacteremia and acute bacterial meningitis.
Occasionally, it causes epiglottitis (obstructive laryngitis), cellulitis,
osteomyelitis, and joint infections. Nontypable H. influenzae
causes ear infections (otitis media) and sinusitis in children,
and is associated with respiratory tract infections (pneumonia) in
infants, children and adults.
Figure 3. Tissues infected by type b and nontypable Haemophilus
influenzae
Seven serotypes of the bacterium have been identified on the basis of
capsular polysaccharides. Until the implementation of widespread vaccination
programs, type b H. influenzae was the most common cause of meningitis
in children between the ages of 6 months and 2 years (see figure below),
resulting in 12,000 to 20,000 cases annually in the U.S. It would be
interesting to view comparative data since the era of vaccination against H.
influenzae meningitis, which began in 1985. Certainly, there are fewer than
100 cases annually of bacterial meningitis caused by H. influenzae type
b.
Figure 4. Age-specific incidence of bacterial meningitis caused by Haemophilus
influenzae, Neisseria meningitidis and Streptococcus pneumoniae
prior to 1985
Disease caused by H. influenzae usually begins in the upper
respiratory tract as nasopharyngitis and may be followed by sinusitis and
otitis, possibly leading to pneumonia. In severe cases, bacteremia may occur
which frequently results in joint infections or meningitis.
Virulence
H. influenzae does not produce any demonstrable exotoxins The direct
role of endotoxin in meningitis or bacteremia is unclear, although
the Gram-negative bacterium's outer membrane lipooligosaccharide is
thought to play a role in inflammation associated with otitis media. All
virulent strains produce neuraminidase and an IgA protease, but
the role of these extracellular enzymes in invasion is unproven. Fimbriae
increase the adherence of bacteria to human mucosal cells in vitro, and they
are required for successful colonization of the nasopharynx. The Anton antigen,
as defined in red blood cells, appears to be the receptor.
Virulence, at least in the case of bacteremia and meningitis, is directly
related to capsule formation. Virtually all of these infections are caused by
the type b serotype, and its capsular polysaccharide, containing ribose,
ribitol and phosphate, is the proven determinant of virulence. The capsule
material is antiphagocytic, and it is ineffective in inducing the alternative
complement pathway, so that the bacterium can invade the blood or cerebrospinal
fluid without attracting phagocytes or provoking an inflammatory response and complement-mediated
bacteriolysis. For this reason, anticapsular antibody, which promotes both
phagocytosis and bacteriolysis, is the main factor in immune defense against H.
influenzae infections (below).
The polyribosyl ribitol phosphate (PRP) capsule is the most important
virulence factor because it renders type b H. influenzae resistant to
phagocytosis by polymorphonuclear leukocytes in the absence of specific
anticapsular antibody, and it reduces the bacterum's susceptibility to the
bactericidal effect of serum. However, susceptibility to the bactericidal
effect of serum depends on the presence of antibodies to a number of other
antigenic sites, including the lipooligosaccharide and outer membrane
proteins designated as P1 and P2.
Type b H. influenzae is plainly the most virulent of the Haemophilus
species; 95 percent of bloodstream and meningeal Haemophilus infections
in children are due to this bacterium. In contrast, in adults, nontypable
strains of H. influenzae are the most common cause of Haemophilus
infection, presumably because most adults have naturally acquired antibody to
PRP.
Immunity
The age incidence of H. influenzae meningitis is inversely
proportional to the titer of bactericidal antibody in the blood, whether
passively acquired from the mother or actively formed (see Figure 5 below).
Without artificial immunization, in children aged 2 months to 3 years, antibody
levels are minimal; thereafter antibody levels increase and the disease becomes
much less common.
From this curve, it is obvious that artificial active immunization should begin
at 2 months of age, when nearly all passive immunity has waned, and the child
enters a vulnerable non immune period of life.
Figure 5. Relation of the age incidence of bacterial meningitis caused by
Haemophilus influenzae to bactericidal antibody titers in the blood (data pre
1985)
H. influenzae is susceptible to lysis by antibody and complement.
Furthermore, anticapsular antibodies promote phagocytosis, as well as
bacteriolysis. Thus, serum antibody, complement, lysozyme and phagocytes can
work in concert during a bacteremia. During meningitis, phagocytosis is
probably the main host defense mechanism since complement rarely occurs in the
cerebrospinal fluid.
For many years it was believed that bactericidal antibody directed against
PRP capsule ofH. influenzae type b was entirely responsible for host
resistance to infection. However, some recent studies have stressed a role for
antibody to somatic (cell wall) antigens as well. For example, antibody to PRP
can often be detected in the sera of children on admission to the hospital with
sepsis due to H. influenzae type b. Adsorption of immune serum with PRP
alone does not remove its protective capabilities, whereas adsorption with
whole organisms does. Separation of the outer membrane of type b H.
influenzae into its many protein constituents reveals several individual
membrane proteins that may be associated with immunity. Bactericidal antibodies
that react with individual outer membrane proteins or with lipooligosaccharide
constituents have been identified. These findings support indicate the
potential importance of antibody to noncapsular antigens in immunity to H.
influenzae type b infection. In addition, opsonizing antibodies, which also
play a role in protection, may be directed against PRP or somatic constituents
(see figure below).
Figure 6. Phagocytic engulfment of H. influenzae bacterium opsonized
by antibodies specific for the capsule and somatic antigen
Recent studies of nontypable H. influenzae have shown that bactericidal
antibody to outer membrane proteins develop in infants in response to otitis
media caused by the organism. Normal adults generally have both bactericidal
and opsonizing antibodies directed against nontypable H. influenzae.
Treatment and Prevention
Virtually all patients treated early in the course of H. influenzae
meningitis are cured. The mortality rate of treated infections is less than 10
percent, but nearly 30 percent of the children who recover have residual
neurologic effects. Ampicillin has been the drug of choice, but presently over
20 percent of all strains of H. influenzae are resistant to ampicillin
because of plasmid-mediated ß-lactamase production.
The recommended treatment for H. influenzae meningitis is ampicillin
for strains of the bacterium that do not make ß-lactamase, and a
third-generation cephalosporin or chloramphenicol for strains that do.
Amoxicillin, together with a substance such as clavulanic acid, that blocks the
activity of ß-lactamase, has been unreliable in treatment of meningitis,
although it is effective in treatment of sinusitis, otitis media and
respiratory infections. Chloramphenicol was long considered the drug of
choice for meningitis caused by penicillin-resistant H. influenzae, and
it is still highly effective, but not without potential toxic side effects.
Third-generation cephalosporins, such as ceftriaxone or cefotaxime, are
effective against H. influenzae and penetrate the meninges well.
Tetracyclines and sulfa drugs remain effective in treating sinusitis or
respiratory infection caused by nontypable H. influenzae. Amoxicillin
plus clavulanic acid (Augmentin) is effective against ß-lactamase producing
strains. Erythromycin is ineffective in treatment ofH. influenzae
infections.
The use of polyribosyl ribitol phosphate (PRP) vaccine and, more recently,
protein-conjugated PRP, has vastly reduced the frequency of infection due to
type b H. influenzae. The PRP vaccine consists of the type b
capsular polysaccharide. Like most bacterial polysaccharides, it elicits a
strong primary antibody response, but with little induction of memory. H.
influenzae type b Hib conjugate vaccines, which couple the
polysaccharide to a protein, induce memory type antibody responses in children
and are effective in younger infants who are at higher risk for the disease.
There are several types of Hib conjugate vaccines available for use.
All of the vaccines are approved for use in children 15 months of age and older
and some are approved for use in children beginning at 2 months of age. All of
the vaccines are considered effective. The vaccines are given by injections.
More than 90% of infants obtain long term immunity with 2-3 doses of the
vaccine.
All children should have a vaccine approved for infants beginning at 2
months. Depending on the type used, the recommended schedule for infants will
vary. All unvaccinated children 15 - 59 months old should receive a single dose
of conjugate vaccine. Children 60 months of age or older and adults normally do
not need to be immunized.
Whether the vaccine provides protection against ear infections is not known.
It also does not protect against diseases caused by other types of Haemophilus.
nor does it protect against meningitis caused by other types of bacteria.
Specific characteristics of the four conjugate vaccines available for
infants and children vary based on the type of protein carrier, the size of the
polysaccharide, and the chemical linkage between the polysaccharide and carrier
(see Table 1 below).
Current recommendations for vaccination of infants require parenteral
administration of three different vaccines, diphtheria-tetanus-pertussis (DTP),
Hib conjugate, and hepatitis B, during two or three different visits to a
health-care provider. TETRAMUNE (see table footnote below) is the first
licensed combination vaccine that provides protection against diphtheria,
tetanus, pertussis, and Hib disease.
Table 1. Hib conjugate vaccines licensed for use among
children
|
Vaccine |
Trade name |
Polysaccharide |
Linkage |
Protein carrier |
|
PRP-D |
ProHIBiT |
Medium |
6-carbon |
Diphtheria toxoid |
|
HbOC* |
HibTITER |
Small |
None |
CRM197 mutant Corynebacterium diphtheriae toxin protein |
|
PRP-OMP |
PedvaxHIB |
Medium |
Thioether |
Neisseria meningitidis outer membrane protein complex |
|
PRP-T |
ActHIB |
Large |
6-carbon |
Tetanus toxoid |
* TETRAMUNE consists of HbOC and DTP vaccine (TRI-IMMUNOL), also
manufactured by Lederle-Praxis.
Tailpiece
Before 1985, Haemophilus influenzae type b (Hib) was the most common
cause of bacterial meningitis in children under 5 years of age (approximately
12,000 cases per year, most in children younger than 18 months). Approximately
5% of affected children died, and neurologic sequelae developed in 15% to 30%
of the surviving children. An additional estimated 7,500 cases of other
invasive Hib infections also occurred annually in young children. The
cumulative risk for Hib invasive disease before the age of 5 was one in 200
children, similar to the risk for poliomyelitis during the 1950s.
In 1985, the first Hib polysaccharide vaccines were licensed for use in the
United States. These vaccines contained purified polyribosylribitol phosphate
(PRP) capsular material from the type b serovar. Antibody against PRP was shown
to be the primary component of serum bactericidal activity against the
organism. PRP vaccines were ineffective in children less than 18 months of age
because of the T-cell-independent nature of the immune response to PRP
polysaccharide.
Conjugation of the PRP polysaccharide with protein carriers confers
T-cell-dependent characteristics to the vaccine and substantially enhances the
immunologic response to the PRP antigen. In 1989, the first Hib conjugate
vaccines were licensed for use among children 15 months of age or older. In
1990, two new vaccines were approved for use among infants.
The incidence of Hib invasive disease among children aged 4 years or younger
has declined by 98% since the introduction of Hib conjugate vaccines. One goal
of the Childhood Immunization Initiative was to eliminate invasive Hib
disease among children aged 4 years or younger by 1996. However, approximately
300 cases of Haemophilus influenzae invasive disease per year continue
to be reported in the U.S., mainly in non immunized children. Most cases
are caused by nontypable Haemophilus influenzae. The bar graph below
(Figure 7) shows the age distribution of cases in 1996 and is comparable to
Figure 5, which displays results from the pre-imuunization era.
Figure
7. Age-specific incidence of bacterial meningitis in children caused by Haemophilus
influenzae in 1996
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Edited on Mar 1, 1999 by Kenneth Todar University of Wisconsin-Madison Department of Bacteriology
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