Gram-Negative Bacteria:

Haemophilus spp and other Fastidious

Gram-Negative
Bacilli
By : DR. E. Andeza
 Introduction
• The HACEK organisms are a group of fastidious Gram-negative bacteria that
are an unusual cause of infective endocarditis, which is an inflammation of
the heart due to bacterial infection.
• HACEK is an abbreviation of the initials of the genera of this group of
bacteria:
• Haemophilus
• Aggregatibacter (previously Actinobacillus) Haemophilus aphrophilus and
Haemophilus paraphrophilus
• Cardiobacterium
• Eikenella
• Kingella
• The HACEK organisms are a normal part of the human microbiota, living in
the oral-pharyngeal region.
 OBJECTIVES
• Identify the many microbiologic features of the organisms
• Identify its diagnostic features
• Diseases caused
 INTRODUCTION
• > Gram negative
• >Cocobacilli bacteria
• > Pleomorphic(variation of morphology or functional
methods of the individual cell, rather than a heritable change )

• Mucous membranes of the upper

respiratory tract
• Mouth
• Vagina
• Intestinal tract
• Species : H. Influenzae , H. ducreyi
Haemophilus influenzae
• Haemophilus influenzae is a Gram-negative, coccobacillary, facultatively
anaerobic capnophilic pathogenic bacterium of the family Pasteurellaceae.
H. influenzae was first described in 1892 by Richard Pfeiffer during an
influenza pandemic.
• He incorrectly described Haemophilus influenzae as the causative microbe,
which retains "influenza" in its name.
• important cause of meningitis in unvaccinated children and causes upper
and lower respiratory tract infections in children and adults
• H influenzae was the most common cause of bacterial meningitis in
children from 5 months to 5 years of age until the early 1990s when the
• conjugate vaccines became available (see later discussion)
Morphology and Identification
• Acute infections:
• the organisms are short (1.5 μm) coccoid bacilli,
sometimes occurring in pairs or short chains.
• In cultures, the morphology depends both on the
length of incubation and on the medium. At 6–8
hours in rich medium, the small coccobacillary forms
predominate. Later, longer rods and very
pleomorphic forms
• young cultures (6–18 hours) on enriched medium
express a definite capsule. The capsule is the antigen
used for “typing” H influenzae
• Chocolate agar: flat, grayish, translucent colonies
with diameters of 1–2 mm are present after 24
hours of incubation.
Haemophilus
• does not grow on sheep blood agar
except around colonies of
staphylococci (“satellite
phenomenon”).
• Staphylococcus aureus produces the V
factor as a metabolic by-product.
Haemophilus species may grow on
sheep blood agar (BAP) very close to
the colonies of S. aureus (as V factor)
• Hemolytic variants :
• H influenzae :Haemophilus haemolyticos
• Haemophilus parainfluenzae:
• Haemophilus parahaemolyticus
Growth
Characteristics
• Growth factors :X and V
• Factor X acts physiologically as hemin;
• Factor V can be replaced by nicotinamide
adenine dinucleotide (NAD) or other
coenzymes
• Carbohydrate fermentation is useful in
species identification as is the presence or
absence of hemolysis
serotyping on the basis of capsular
polysaccharides
• H influenzae and H
parainfluenzae can be biotyped
on the basis of the production of
1.indole
2.ornithine decarboxylase
3.urease.

• invasive infections caused by H

influenzae belong to biotypes
• I and II (there are a total of
eight)
Virulence Factors
• Capsule
• Encapsulated H. influenzae contains capsular polysaccharides of one of six
types (a-f). Capsule protects Hib from host immune functions and known
plays a major role for its invasiveness.
• The capsular antigen of type b is a polyribosyl ribitol phosphate (PRP). The
capsule is antiphagocytic and one of the major virulence factors of H.
influenzae. PRP facilitates invasion of the bloodstream and spread of
infection by the hematogenous route.
Antigenic Structure

• Encapsulated H influenzae contains capsular

polysaccharides (molecular weight >150,000) of
one of six types (a–f).
• The capsular antigen of type b is a polyribitol
ribose phosphate (PRP). Encapsulated H
influenzae can be typed by slide agglutination,
coagglutination with staphylococci, or
agglutination of latex particles coated with
type-specific antibodies.
• A capsule swelling test with specific antiserum
is analogous to the quellung test for
pneumococci.
Typing
• Typing can also be done by
immunofluorescence.
• Most H influenzae organisms in the
normal microbiota of the upper
respiratory tract are not
encapsulated and are referred to as
nontypeable (NTHi).
• NTHi tends to cause chronic
bronchitis, otitis media, sinusitis,
and conjunctivitis
• The somatic antigens of H influenzae
consist of outer membrane proteins.
Lipooligosaccharides (endotoxins)
share many structures with those of
neisseriae
Virulence Factors
• Adhesion proteins
• Adhesin proteins such as HMW1
and HMW2 mediate attachment to
the human epithelial cells in the
airway. Adhesion proteins play
major roles in the attachment of
nontypeable Haemophilus
influenzae (NTHi) to the respiratory
epithelium which in turn promotes
colonization and subsequent
epithelial and endothelial invasion
by this bacteria.
Pathogenesis
Lipooligosaccharides (LOS)
• H. influenzae produces no exotoxins. The lipooligosaccharide is the endotoxins that plays role in
the attachment and invasiveness of this organism. Paralysis of the ciliated respiratory epithelium
by LOS thwart its mechanical clearance as a part of a non-specific host defense mechanism. In
NTHi stains, LOS is a critical factor for defense against host complement.
• The polyribose phosphate capsule of type b H influenzae is the major virulence factor

Pili
• Pili and the major outer membrane P2 protein bind sialic acid-containing moieties on epithelial
cell surfaces

• Outer membrane proteins

• They contribute in adhesion and invasion of host tissues.
Virulence factors
• IgA1 protease
• H. influenzae produces IgA1protease which cleaves immunoglobulin A
at the hinge region. The activity of IgA1 protease is thought to
prevent agglutination at the mucosal surface and subsequent
mechanical clearance of the pathogen. Increased levels of IgA
protease activity correlated with increased invasiveness of NTHi
strains in humans
Phase variation

• Phase variation helps in immune

evasion by the modification of
outer surface protein of the
pathogen which helps the
pathogen to adapt to changes in
the host environment.
Hemoglobin bind proteins
(HgbA, HgbB, HgBC) and
adhesins (OapA, HMW1, HMW2)
etc are shown to undergo phase
variation.
Hemophilus : Virulence
• Encapsulated strains of
Haemophilus influenzae are
further classified into 6 different
serotypes (a-f) based on the
presence of capsular
polysaccharide, with the
Haemophilus influenzae
serotype b (Hib) being the most
virulent strain. In spite of the
name, H. influenzae do not
cause influenza (the flu)
What are the virulence factors of Haemophilus
influenzae?
• Haemophilus influenzae has
several virulence factors that
play a crucial role in patient
inflammatory response.
• Major virulence factors of H.
influenzae are: Encapsulated H.
influenzae contains capsular
polysaccharides of one of six
types (a-f).
Hemophilus Influenzae
Treatment
• Many strains of H influenzae type b • Essentially all strains are susceptible
are susceptible to ampicillin, but up to to third-generation cephalosporins
25% produce a β-lactamase under and carbapenems. Cefotaxime given
control of a transmissible plasmid and intravenously gives excellent results.
are resistant. E Prompt diagnosis and antimicrobial
therapy are essential to minimize late
neurologic and intellectual
impairment.
• Prominent among late complications
of H influenzae type b meningitis
• is the development of a localized
subdural accumulation of fluid that
requires surgical drainage.the
Epidemiology, Prevention, and Control
• Encapsulated H influenzae type b is • series consists of three doses at 2, 4, and 6
transmitted from person to person by the months of age or two doses given at 2 and 4
respiratory route. months of age.
• H influenzae type b disease can be prevented • An additional booster dose is given sometime
by administration of Haemophilus b conjugate between 12 and 18 months of age
vaccine to children.
• three PRP polysaccharide-protein
monovalent conjugate vaccines
• (polysaccharide linked to outer membrane
protein complex) available for use in the
United States: PRP-OMP (PedvaxHIB,
• Merck and Co., Inc.), PRP-T (ActHIB, Sanofi
Pasteur, Inc.), and PRP-T (Hiberix,
GlaxoSmithKline).
HAEMOPHILUS AEGYPTIUS
• was formerly called the Koch-
Weeks bacillus
• associated with highly
communicable form of
conjunctivitis (pinkeye) in children.
• closely related to H influenzae
biotype III, the causative agent of
Brazilian purpuric fever. The latter
is a disease of children
characterized by fever, purpura,
shock, and death. In the past,
• these infections were mistakenly
attributed to H aegyptius
HAEMOPHILUS DUCREYI

• Haemophilus ducreyi is a fastidious gram-negative

coccobacillus bacteria.

• It causes the sexually transmitted disease chancroid, a

major cause of genital ulceration in developing countries
characterized by painful sores on the genitalia.
• Chancroid starts as an erythematous papular lesion that
breaks down into a painful bleeding ulcer with a necrotic base
and ragged edge.
• More recently, it has also been found to cause chronic skin
ulceration away from the genitalia, infect children and adults,
and behave in a manner that mimics yaws. These strains seem
to have diverged quite recently.
Hemophilus ducreyi
• The small, gram-negative rods occur in strands in the
lesions, usually in association with other pyogenic
microorganisms.
• H ducreyi requires X factor but not V factor. It is
grown best from scrapings of the ulcer base that are
inoculated onto chocolate agar containing 1%
IsoVitaleX and vancomycin, 3 μg/mL; the agar is
incubated in 10% CO2 at 33°C.
• Nucleic acid amplification methods are more
sensitive than culture.
• There is no permanent immunity after chancroid
infection
• >Unlike other Haemophila, H. ducreyi is unable to synthesize
heme because it lacks the enzyme ferro-chelatase, which is used
to catalyze the synthesis of heme by inserting ferrous iron into
protoporphyrin IX.
• Its main source of heme is from hemoglobin, which is
observed to be acquired through cell invasion of the host.
• The process by which H. ducreyi takes in the hosts heme has
been shown to be a "TonB- and TdhA-independent mechanism,
possibly diffusion" (Thomas et al, 1998).
• fimbria like protein (flp) must be present for virulence.

• Some important molecules that are secreted by H. ducreyi

include
>cytolethal distending toxin (CDT)
> hemolysin (Cope et al, 1997).
• CDT is also secreted and could be the cause of the generation
and slow healing of ulcers ( The bacterium also causes the host to
secrete Interleukin 8 (IL-8) and IL-6, potent chemoattractants for
neutrophils.
• Virulence determinants have been difficult to identify (
signature-tagged mutagenesis are being researched for properly
identifying determinants
Capnophiles requiring 5-10% CO2
• Capnophiles are microorganisms that thrive in the presence of high
concentrations of carbon dioxide (CO 2). requiring 5-10% CO2
• Some capnophiles may have a metabolic requirement for carbon
dioxide, while others merely compete more successfully for resources
under these conditions
• Examples :
• Haemophilus influenzae
• Neisseria gonorrhoeae
• Aggregatibacter
• Kingella
• Cardiobacterium
Haemophilus
Aegypticus
• Haemophilus aegypticus
(Haemophilus influenzae
biogroup aegyptius), a
species of the genus
Haemophilus in the family
Pasteurellaceae, is an
immotile, Gram-negative,
coccoid rod bacterium.
 Haemophilus
Aphrophilus
• Haemophilus aphrophilus
was a species of Haemophilus,
but the name has recently
been changed to
Aggregatibacter aphrophilus .
It is one of the HACEK
organisms.
 Haemophilus
Haemolyticus
• Haemophilus haemolyticus is a species of
gram-negative bacteria that is related to . H.
haemolyticus is generally nonpathogenic,
however there have been two cases of
H.haemolyticus causing endocarditis. There is
active research on H. haemolyticus especially
in taxonomy and in identification, e.g.
Haemophilus
Parahaemolyti
cus
• Haemophilus
parahaemolyticus
Hae·moph·i·lus
par·a·hae·mo·ly't·i·cus a
bacterial species found
in the upper respiratory
tract and associated
frequently with
pharyngitis; occasionally
causes subacute
endocarditis.
Aggregatibacter aphrophilus
• H aphrophilus and H paraphrophilus have been combined into a
single genus and species, A aphrophilus.
• Other members of the Aggregatibacter genus include
• A actinomycetemcomitans
• A segnis.
• These organisms are associated with a variety of infections including
endocarditis.
C. hominis
• C. hominis is a catalase-negative,
oxidase-positive, indole-
producing, Gram-negative rod.
• Its morphology has classically
been described as highly
pleomorphic and irregularly
staining, although homogeneous
bacilli with uniform shapes may
be seen with the addition of
yeast extract
Cardiobacterium hominis
• C. hominis has been sensitive to penicillin and
penicillin derivatives such as ampicillin.
• penicillin-resistant strains, including those that
produce beta-lactamases
• Clinical guidelines thus recommend that C.
hominis and other HACEK organisms be presumed
to harbor ampicillin resistance and therefore be
treated with a third-generation cephalosporin.
• C. hominis and other HACEK organisms also
exhibit in vitro susceptibility to trimethoprim-
sulfamethoxazole, fluoroquinolones, and
aztreonam.
• C. hominis is often resistant to erythromycin.
• cefotaxime use may be not appropriate for C.
hominis endocarditis, an alternative regimen might
include association of co-amoxiclav and gentamicin
Eikenella
• The acronym stands for Haemophilus, Aggregatibacter, Cardiobacterium,
Eikenella, and Kingella: all HACEK members are fastidious Gram-negative
bacteria associated with infective endocarditis
MICROSCOPIC APPEARANCE

Size: 0.3-0.4
Gram Stains: Morphology: micrometers by 1.5-4.0
Negative. Straight rods. micrometers with
rounded ends.

Motility:They are non-

motile but may exhibit
a "twitching motility" Spores: Non-
Capsules: None.
on some media. Short sporeforming.
filaments are
occasionally formed.

Other: Hemin is
usually required for
growth under aerobic
conditions.
MACROSCOPIC APPEARANCE
Colonies are 0.2-0.5mm at 24 hours; 0.5-1.0mm at 48 hours. Colonies have moist,
clear centers surrounded by flat, spreading growth. May appear to "corrode" the
surface of the agar medium. A slight yellow pigmentation may be present in older
cultures. Plate cultures have an odor described as "bleach like" or as resembling that
of Haemophilus and Pasteurella species. Growth in liquid media is usually poor.
METABOLIC PROPERTIES
Facultatively anaerobic. No acids formed from glucose or other carbohydrates.
KEY BIOCHEMICAL REACTIONS
Oxidase-positive.
Nitrates reduced to nitrite.
Catalase-negative.
Urease-negative.
Indole-negative.
Hydrolyzes hippurate.
Lysine-decarboxylase-positive.
Arginine-dihydrolase-negative.
HABITAT
Eikenella corrodens is commonly found in the human mouth and intestine, and has
also been isolated from the genitourinary tract.
PATHOGENICITY
Eikenella corrodens is an opportunistic pathogen, particularly likely to produce
infection in compromised hosts. It has been implicated in osteomyelitis, arthritic, respiratory,
and septicemic infections. Infection related to the mouth and upper and lower respiratory
tract sinuses, lips and face is not uncommonly reported. Serious infections including brain
abscesses have sometimes been encountered. Transfer from the mouth may cause infection
of human bites. The ability of E. corrodens to survive in the intestine leads to its presence in
abdominal infections, including wound infection, liver and other abscesses as well as
peritonitis. Meningitis and fatal endocarditis have also been reported. (2)

RECOMMENDED MEDIA
For culture: Columbia Blood Agar, Blood Agar 5%.
For selective isolation: Todd Hewitt Agar with Clindamycin (5mcg/ml), Potassium Nitrate
(2mg/ml) and Hemin (5mcg/ml). (1) (Not available commercially.)
For maintenance: Columbia Blood Agar, Blood Agar 5%. Horse serum is recommended for
long-term storage at -70 degrees C.
Optimum pH: 7.3.
INCUBATION
Temperature: 35 degrees C. in 5-10% CO 2 .
Time: 24-48 hours.
Atmosphere: Aerobic; facultatively anaerobi
 Kingella
Kingella kingae is a common etiology of pediatric bacteremia and the
leading agent of osteomyelitis and septic arthritis in children aged 6 to 36
months.
>Gram-negative bacterium is carried asymptomatically in the oropharynx
and disseminates by close interpersonal contact.
The colonized epithelium is the source of bloodstream invasion and
dissemination to distant sites, and certain clones show significant
association with bacteremia, osteoarthritis, or endocarditis.
Kingella kingae produces an RTX (repeat-in-toxin) toxin with broad-
spectrum cytotoxicity that probably facilitates mucosal colonization and
persistence of the organism in the bloodstream and deep body tissues.
With the exception of patients with endocardial involvement, children with
K. kingae diseases often show only mild symptoms and signs, necessitating
clinical acumen.
> isolation of K. kingae on routine solid media is suboptimal, and detection
of the bacterium is significantly improved by inoculating exudates into
blood culture bottles and the use of PCR-based assays.
> is generally susceptible to antibiotics that are administered to young
patients with joint and bone infections.
➢ β-Lactamase production is clonal, and the local prevalence of β-
lactamase-producing strains is variable.
➢ adequately and promptly treated, invasive K. kingae infections with no
endocardial involvement usually run a benign clinical course.
Brucella
Brucella

• Brucella is a genus of Gram-

negative bacteria, named after
David Bruce (1855–1931).
• They are small (0.5 to 0.7 by 0.6 to
1.5 µm), non encapsulated, non
motile, facultatively intracellular
coccobacilli .
• Brucella spp. are the cause of
brucellosis, which is a zoonosis
transmitted by ingesting
contaminated food
• Highly fastidious
Diagnosis
• Milk ring test
• agglutination test is used to
identify Ig G
 Francisella
• Francisella is a genus of pathogenic,
Gram-negative bacteria. They are small
coccobacillary or rod-shaped, nonmotile
organisms, which are also facultative
intracellular parasites of macrophages.
Strict aerobes, Francisella colonies bear
a morphological resemblance to those of
the genus Brucella
 • The subspecies of F. tularensis: F. tularensis
subspecies of F. subsp. tularensis (Type A), which is the most
common type in North America, and is highly
tularensis virulent in humans and animal
Symptoms depend on the type of Tularemia
Ulceroglandular tularemia
Skin ulcer on the site of infection
Painful and swollen lymph glands
• Chills
• Fever
• Exhaustion
• Headache
• Glandular tularemia
Symptoms are similar to ulcero glandular tularemia but without skin ulcers
• Oculoglandular tularemia
Eye redness
• Eye pain
• Discharge and swelling of the eyes
• Ulcer inside the eyeli
Francisella tularensis
• is a fastidious bacteria which
requires enriched medium for
growth
• is a pathogenic species of Gram-
negative coccobacillus, an aerobic
bacterium.
• It is nonspore-forming, nonmotile,
and the causative agent of
tularemia, the pneumonic form of
which is often lethal without
treatment. It is a fastidious,
facultative intracellular bacterium
Naturally occurring tularemia is a zoonotic disease caused by the bacterium
Francisella tularensis, which is a hardy organism capable of surviving for weeks at low
temperatures in water, moist soil, hay, straw, or decaying animal carcasses.

There are 4 subspecies of F. tularensis: F. tularensis subsp. tularensis (Type A), which
is the most common type in North America, and is highly virulent in humans and
animals. F. tularensis subsp. Holarctica (Type B), a less virulent type, responsible for
human tularemia infection in Europe and Asia as well as North America; F. tularensis
subsp. novocida, another less virulent type; and F. tularensis subsp. mediaasiatica,
which is also of low virulence.

Small mammals such as voles, mice, squirrels, and rabbits are natural reservoirs for
F. tularensis. These animals acquire tularemia through bites from ticks, fleas, and
mosquitoes and also through contact with contaminated environments. Naturally
acquired human infection can occur through bites from infected arthropods (usually
ticks); contact with infected animal tissues or fluids; direct contact with or ingestion of
contaminated water, food, or soil; or inhalation of aerosolized bacteria. Naturally
acquired human infection tends to occur predominately in rural areas. F. tularensis is
so infectious that simply opening a laboratory culture plate without adequate
protective equipment can lead to infection.
 Pathogenesis:
The type species, F. tularensis, causes the disease tularemia or
rabbit fever.[4] F. novicida and F. philomiragia (previously
Yersinia philomiragia) are associated with sepsis and invasive
systemic infections.
Laboratory characteristics
Francisella species can survive for several weeks in the environment;
Microscopic image of F. tularensis. paradoxically, they can be difficult to culture and maintain in the
Source: National Institute of Allergy and laboratory.
Infectious Disease (NIAID) Laboratory of Growth is slow (though increased by CO2 supplementation) and the
Intracellular Parasites, Tularemia organisms are fastidious, with most Francisella strains requiring
Pathogenesis Section. cystine and cysteine media supplementation for growth. Growth has
been successful on several media types, including chocolate agar
and Thayer–Martin medium with appropriate additives as noted
above. Attempted isolation on MacConkey agar is not reliable or
generally successful.[4]

After 24 hours of incubation on appropriate solid media, Francisella

colonies are generally small (1 to 2 mm), opaque, and white-gray to
bluish-gray in color. Colonies are smooth, with clean edges and,
after a 48 hours of growth, tend to have a shiny surface.
• Most Francisella strains requires the following media
supplementation for growth
• . Cystine
• . Cysteine