Download Haemophilus and Acid-fast bacteria Haemophilus General

Dr. Manal Al khulaifi
Reem ALjowaie
Haemophilus and Acid-fast bacteria
 Haemophilus
o The term Haemophilus represents a large group of Gram-negative rods that like to
grow on chocolate blood agar and restricted to organisms that are dependent on one
or both of certain growth promoting substances termed X (haematin) and V (NAD)
which present in blood; therefore these organisms grow only on blood agar or
chocolate agar.
 General characters:
1. Non intestinal pleomorphic Gram negative coccobacilli
2. Grown under aerobic conditions or under slight CO2 tension (5% CO2)
3. Non motile
4. Non spore forming
5. Usually capsulated (poly ribitol phosphate)
6. Oxidase and catalase positive
7. Fastidious bacteria (mostly)
8. Facultative anaerobic.
 Species of Haemophilus
o H. influenza
o H. ducreyi
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H. aegypticus
H. parainfluenzae
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REEM ALJOWAIE
Dr. Manal Al khulaifi
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H. influenza
 Serology
o Heamophilus influenzae are divided according to ( capsular antigens) into six
serogroups (a through f) . The majority of strains that cause meningitis belong to
invasive serogroup b .
o Most of the strains that cause chronic bronchial disease are non-capsulated .
- Non capsulated strains colonize the nasopharynx in up to 80% of healthy
individuals in the upper respiratory tract and become a secondary pathogen on
respiratory mucosa that has become susceptible to bacterial attack after primary
influenzae virus infection.
-
Non capsulated strains are less invasive, but they are apparently able to induce
an inflammatory response that causes disease.
-
It may spread locally and cause pneumonia, otitis media, and sinusitis.
-
Encapsulated strains, are a very minor colonized in 5% of healthy individuals in
the upper respiratory tract.
-
Encapsulated organisms can penetrate the epithelium of the nasopharynx and
invade the blood capillaries directly.
-
Their capsule allows them to resist phagocytosis.
- It cause pediatric meningitis, otitis media & epiglottitis (obstructive laryngitis).
 Laboratory Features
 Specimens
These include cerebrospinal fluid (c.s.f) , nasopharyngeal specimens , pus, and blood
for culture.
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Dr. Manal Al khulaifi
Reem ALjowaie
 Morphology
- H.influenzae is a small , non-motile , Gram negative , coccobacillus or short rod.
- Long thread-like and pleomorphic forms may be seen in c.s.f (with pus cells , or
following culture.
- The capsule which surrounds capsulated strains can be demonstrated by using specific
antiserum which added to a slide of the organism allows swelling of the bacterial
capsule thus permitting rapid diagnosis of H. influenzae in sputum.( Capsule swelling)
 Culture
- H.influenzae grows aerobically and only poorly anaerobically .
- Growth is best in a moist carbon dioxide enriched atmosphere (5% CO2)
- The optimum growth temperature is 35-37 °C .
- Media used to grow H.influenzae must contain haemin or other iron-containig
prophyrin and nicotinamide adenine dinucleotide (NAD) or its phosphate (NADP).
The porphyrin requirement is referred to as growth factor X and the NAD or NADP
requirement as growth factor V .
- Factor X is used by H.influenzae to produce essential respiratory enzymes such as
cytochromes , catalase and peroxidase .While , factor V is used as an electron carrier
in the organism’s oxidation –reduction system .
- On Blood agar: A 24 h colony of H. influenzae on blood agar is very small usually
non hemolytic ( contain less V factor )
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REEM ALJOWAIE
Dr. Manal Al khulaifi
Reem ALjowaie
- On chocolate agar: A 24 h colony of H. influenzae on chocolate agar is larger than
that observed on blood agar. So , its grows well on chocolate agar because it contains
factors X and V .
Haemophilus influenzae as seen using a Gram-stain technique.
Cerebrospinal fluid culture positive for Haemophilus influenzae, type b (Gram stain)
 PHENOMENON: Satellitism Bacteria
- Blood agar contains much X factor and little V factor and so the presence of another
microorganism such as Staph. aureus which produces V factor will support the
growth of H. influenzae i.e. larger colonies of H. influenzae are observed near Staph.
aureus colonies.
- The “help” given by Staph. aureus ,form the basis of the satellitism test which is a
simple way of recognizing H. influenzae .
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Dr. Manal Al khulaifi
Reem ALjowaie
- S.pneumoniae also produces factor V and causes H. influenzae to show satellitism .
PHENOMENON: Satellitism Bacteria
 Identification of H.influenzae using X, V , XV tablets or discs
- H.influenzae can be identified by its requirement for factors X and V using factor
identification tablets
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REEM ALJOWAIE
Dr. Manal Al khulaifi
Reem ALjowaie
H. parainfluenzae
 H. parainfluenzae is a commensal of the upper respiratory tract Occasionally it causes
respiratory infections, endocarditis, pyogenic arthritis and conjunctivitis , often as an
opportunistic pathogen .
 H. parainfluenzae is differs from H. influenzae in requiring only factor V for its
growth and not factor X .On chocolate agar it forms larger colonies than H.influenzae.
Acid-fast bacteria
 Acid fast bacteria include the Mycobacterium and few species of Nocardia.
 The Mycobacterium include 2 species:
o Mycobacterium tuberculosis, which causes tuberculosis
o Mycobacterium leprae, which causes leprosy
 Mycobacterium contains 40% lipid content in their cell envelop.
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REEM ALJOWAIE
Dr. Manal Al khulaifi
Reem ALjowaie
 High lipid content make it difficult to stain by ordinary dye but requires special dye
as Carbol fuchsin and heating, and once stained are difficult to decolorize with acidalcohol mixture.
 Acid fastness is due to high lipid content of cell envelop.

Laboratory Features
 Specimens
- Sputum , not slaiva is required to detect AFB (acid fact bacilli ). Also, cerebrospinal
fluid is required to investigate tuberculous meningitis.
 Morphology
- M.tubercluosis is a non-sporing , non-capsulated straight or slightly curved slender
rod , measuring 1-4µm x 0.2-0.6µm .
- Acid-alcohol fast (Acid fast stain) using Ziehl-neelsen staining technique .
- Mycobacteria does not gram stain due to its waxy surface , the organism has a Grampositive cell wall .
- Non-motile, Non-capsulate
- Obligate aerobic, Catalase positive
- Most Mycobacteria are found in habitats such as water or soil. However, a few are
intracellular pathogens of animals and humans.
 Ziehl Neelsen Acid fast Stain method
- When stained by the Ziehl-neelsen technique, M.tuberculosis is acid fast and stain
red. This due to mycolic acids (fatty acid ) in the cell wall which form acomplex with
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Dr. Manal Al khulaifi
Reem ALjowaie
carbol fushin (an arylmethane dye) and cannot be removed by the acid alchol in the
decolorizing reagent. After decolorization, methyelene blue is added to organisms to
counterstain any material that is not acid fast.
 Mycobacteria are the third most resistant form of microorganisms, falling just below
prions and endospores of bacteria, generally resistant to drugs and chemical biocides.
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REEM ALJOWAIE
Dr. Manal Al khulaifi
Reem ALjowaie
 Culture
- M.tuberculosis will grow aerobically on a protein enriched medium e.g. Lowenstein
Jensen medium at 35-37 C, which is made by addition of malachite green & some
antibiotics which inhibit the growth of fungi and other bacteria.
- Presence of CO2 enhances the growth.
- M.tuberculosis produces raised, dry, cream (buff) coloured colonies .Visible colonies
are usually produced 2-3 weeks after incubation, Slow growing organism, generation
time each 20 h, but cultures should be incubated for up to 6 weeks before being
discarded.
 Bactec rapid radiometric culture system
- Bactec is an automated early detection system in which specimens are cultured in a
liquid medium containing C14-labelled palmitic acid.
- Growing mycobacteria utilize the acid , releasing radioactive carbon dioxide which is
measured in the Bactec instrument.
- Growth of M.tuberculosis can be detected within 12 days, even before colonies can be
seen.
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REEM ALJOWAIE
Dr. Manal Al khulaifi
Reem ALjowaie
 Nucleic acid probes and nucleic acid amplification tests are used to amplify
specific DNA or RNA sequences extracted from Mycobacterial cells , are used to
detect M.tuberculosis directly in sputum .
 Pathogenesis of TB:
o Primary tuberculosis:
-
The first exposure to M. tuberculosis is called primary tuberculosis
-
TB is a highly communicable disease that is transmitted mainly by droplets due to M.
tuberculosis causing pulmonary infection or by ingestion of cow milk infected with M.
bovis resulting in intestinal infection
- Primary tuberculosis may occur at any epithelial site but common in lung
o Secondary or Reactivation infection:
-
This is usually caused by endogenous TB that have survived in the primary lesion in
less than 5% of those infected and rarely due to exogenous infection
-
Reactivation is seen primarily in immuno-compromised patients
- It is characterized by chronic tissue lesions, formation of tubercles, caseation and
fibrosis
-
Reactivation lesions occur at apex of lung and other well oxygenated organs such as
brain, kidney.
 Clinical manifestations:
Fever, fatigue, night sweats, and weight loss are common. Cough and bloody sputum
are common in pulmonary TB.
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REEM ALJOWAIE
Dr. Manal Al khulaifi
Reem ALjowaie
 Tuberculin test (PPD test) or Mantoux test :
- This is a skin test which is useful in diagnosis of TB specially during the primary
infection where the majority of cases are symptom less
- During primary infection the host acquires hypersensitivity to TB and becomes
positive to tuberculin.
- It is carried out by intradermal injection of 0.1ml of purified protein derivative
(tuberculin) in the forearm.
- The reaction is read by measuring the diameter of induration (palpable raised,
hardened area) across the forearm (perpendicular to the long axis) in millimeters. If
there is no induration, the result should be recorded as "0 mm". Erythema (redness)
should not be measured.The best time for reading is 72 hours after injection.
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The results of this test must be interpreted carefully. The person's medical risk factors
determine at which increment (5 mm, 10 mm, or 15 mm) of induration the result is
considered positive.A positive result indicates TB exposure.
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5 mm or more is positive in
o An HIV-positive person
o Persons with recent contacts with a TB patient
o Persons with nodular or fibrotic changes on chest X-ray consistent with old healed TB
o Patients with organ transplants, and other immunosuppressed patients
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10 mm or more is positive in
o Recent arrivals (less than five years) from high-prevalence countries
o Injection drug users
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Dr. Manal Al khulaifi
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o Residents and employees of high-risk congregate settings (e.g., prisons, nursing
homes, hospitals, homeless shelters, etc.)
o Mycobacteriology lab personnel
o Persons with clinical conditions that place them at high risk (e.g., diabetes, prolonged
corticosteroid therapy, leukemia, etc.,)
o Children less than four years of age, or children and adolescents exposed to adults in
high-risk categories
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15 mm or more is positive in
Persons with no known risk factors for TB
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