Acid-Fast Bacilli

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Acid-FastBacilli
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Mycobacterium Group
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• Definition
• Generally, these microorganisms are
  characterized by their special reaction to stain.
  
• They are difficult to be stained by ordinary
  stain due to the high content of lipoid
  substances in the cell wall.
• However, once they stained they resist
  decolorization even with acid, thus they called
  acid fast bacilli when stained with Zhiel
  -Neelsen stain.

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• The acid fastness is due to the high lipid
  content (about 60) of their cell wall.
• They contain N-glycolylmuramic (mycolic) acid
  instead of N-acetylmuramic acid.
• They are Gram ve but stained poorly.
• Non spore forming, non motile and
  non capsulated.
• This group includes both pathogenic and
  non pathogenic bacteria.

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Classification
Mycobacterium tuberculosis
Mammalian type

• Human.
• Bovine.
• Murin type.

M. leprae
Atypical Mycobacterium
Avian type
Reptilian or cold blood type
Saprophytic Mycobacterium
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Mycobacterium tuberculosis(Human or
Bovine type)
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• Morphology
• Acid Fast beaded bacilli arranged in bundles.
• Non spore forming.
• Non motile.
• Non capsulated.
• Culture characteristics
• Require Dorsets egg media.
• Can grow on in presence of Malachite green
  (Löwenstein-Jensen media) as selective agent.

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• Strictly aerobic.
• Grow very slow, thus require 6-8 weeks before
  discarding the culture as negative.
• Biochemical Reactions
• Niacin test
• Positive in human type.
• Negative in bovine type.

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• Epidemiology
• Tuberculosis still remains among the most
  important communicable diseases in the world
  today with an estimated 50 million active cases
  of who 3 million will die annually from its
  effects.
• In Egypt, the prevalence rate is about 1.7 of
  which two thirds are active cases. The death rate
  is about 15/100,000.

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• There are mainly two modes of infection either
  by droplet (airborne)
  infection and this is generally by human type or
  by consumption of contaminated milk or milk
  products and this is usually by bovine type.
• ?Human type causes pulmonary tuberculosis
  mainly by droplet infection from an open case.
• ?Bovine type is attracted by the ingestion of
  contaminated milk or milk products.

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• The bacilli will reach to the cervical glands.
  Also, the organism may reach to the mesenteric
  lymph nodes causing lymphadenitis.
• From the lymph glands the bacilli may reach to
  many organs causing generalized miliary
  tuberculosis.
• Primary tuberculosis may occur at any epithelial
  site (at any part of the body) however, it is
  most commonly restricted to the lung TB.

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• Pathogenicity
• Mycobacteria survive after ingestion by
  macrophages and behave as
  facultative intracellular organisms.
• The infected cells express histocompatibility
  complex (MHC)-associated bacterial peptides that
  trigger T cells responses.
• Activated CD4 and TH1 cells release large
  amounts of interferon-? (IFN-?), which activates
  the infected macrophages.

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• The activated macrophages, in turn, destroy the
  intracellular Mycobacterium.
• The major pathogens are Mycobacterium
  tuberculosis, the causative agent of
  tuberculosis, and Mycobacterium leprae, the cause
  of leprosy.
• Atypical mycobacterium, such as Mycobacterium
  avium-intracellulare complex and Mycobacterium
  kansasii, can cause tuberculosis disease but less
  frequent pathogens.

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• Rapidly growing mycobacteria, such as
  Mycobacterium chelonei, are saprophytes that
  occasionally cause human disease in
  immunocompromised hosts.
• In human, the tuberculosis is either
• ? Pulmonary type.
• ? Extra-pulmonary type.

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• Determinants of Pathogenicity
• Cording factor is a glycolipid derivative of
  mycolic acid that is present on the outer surface
  of M. tuberculosis. The glycolipid inhibits
  migration of polymorphonuclear (PMN) leucocytes
  and elicits granuloma formation. Also, it is
  immunogenic.
• Sulfatides permits the bacteria to survive in the
  macrophages.
• Antibacterial resistance by mutation.

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• Clinical picture
• The typical symptoms include fever, fatigue,
  night sweats, and weight loss.
• Many organs can be involved. Miliary tuberculosis
  (spread via blood) is characterized by multiple
  disseminated lesions that resemble millet seeds.
  
• Tuberculous meningitis and tuberculous
  osteomyelitis are important
  disseminated forms.

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• Laboratory Diagnosis
• ?Direct Methods
• The specimen.
• Direct Examination (Zhiel-Neelsen stain).
• ? Methods Depend on Isolation
• Culture.
• Biochemical reactions.
• Animal inoculation.
• ?Methods other than isolation
• Tuberculin (Mantoux) Test.
• Chromatographic Analysis.
• Molecular Methods.

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• ?Direct Methods
• The specimen
• Sputum in case of pulmonary tuberculosis.
• Pus in case of skin type.
• Urine in case of urinary tuberculosis.
• CSF or blood in case of meningitis.
• Blood in case of miliary tuberculosis.
• Stool in case of intestinal tuberculosis.

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• Special Remarks
• For sputum It's better to take the morning
  sample and for 3 successive days. The sample
  requires decontamination process and may be
  concentration.
• Pus could be treated as sputum.
• Urine is treated as above and in direct
  examination acid and alcohol must be used to
  avoid the false ve result due to presence of the
  saprophytic mycobacterium in the smegma.

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• CSF it's a normal sterile liquid, thus direct
  examination and culture are done directly from
  the deposit after centrifugation without the need
  of decontamination process.
• Stool treated as urine.

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• Direct Examination
• A film is prepared and suspected specimen and is
  stained with Ziehl - Neelsen stain.
• The appearance of acid-fast bacilli having the
  morphology of TB (tubercle bacilli) is almost
  diagnostic of tuberculosis infection.
• Alternatively, the bacilli could be detected
  with auramine stain and visualized by
  fluorescence microscope.

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• ? Methods Depend on Isolation
• Culture
• May be positive even when the direct test is
  negative and this could be attributed to the
  presence of very few numbers of bacilli in the
  sample.
• The sample (from non sterile sites) is undergone
  a process of decontamination and concentration.

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• Culture is done on Löwenstein-Jensen or
  Middlebrook medium and incubates aerobically at
  37? C and examined weekly up to 8 weeks before
  considering it negative.
• Colonies are dry and warty appearance and buffy
  in color (human type) or soft and flat colonies
  (bovine type).
• Alternatively, culture could be made in liquid
  BACTEC medium, in which radioactive metabolites
  are used and growth can be detected by the
  production of radioactive CO2 in shorter time.

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• Biochemical reactions
• Niacin test is ve in case of human type and -ve
  in case of bovine type.
• Catalase -ve.
• Animal inoculation
• The prepared sample for culture may also be
  injected into two guinea pigs. One of these
  animals is killed after 4-6 weeks and examined
  for the typical lesions of tuberculosis and also
  by film stain and culture. The other animal is
  left under observation.

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• ?Methods other than isolation
• Tuberculin (Mantoux )Test
• The immunological base of this test is the type
  IV hypersensitivity or delayed type which depends
  on cell mediated immune reaction.
• This test was firstly described by Koch who used
  the crude extract of TB (tubercle bacilli) known
  as Koch's Old Tuberculin (K.O.T).
• Recently, a purified protein derivative (PPD) is
  used instead.

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• Tuberculin test is performed by intradermal
  injection of the PPD (5 IU) in one forearm.
• In case of ve tuberculin, a red and indurated
  area (10 mm in diameter) appears after 2-3 days.
  The induration is very important.
• Significance of tuberculin test
• Tuberculin ve means a case or previously
  exposed to TB or vaccinated. Since by adulthood
  as many as 80 of Egyptians are
  positive, thus the test is of great diagnostic
  value (together with the clinical symptoms) in
  childhood.

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• Tuberculin -ve means neither a case nor having
  immunity.
• Tuberculin test is used before BCG vaccination,
  which should be given only to tuberculin -ve
  people. If it is given to tuberculin ve
  individual, it may provoke a virulent
  unwanted reaction.

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• Bacillus CalmetteGuérin is a vaccine against
  tuberculosis that is prepared from a strain of
  the attenuated (weakened) live bovine
  tuberculosis bacillus, Mycobacterium bovis, that
  has lost its virulence in humans by being
  specially subculture in an artificial medium for
  13 years, and also prepared from Mycobacterium
  tuberculosis.

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Albert Calmette
Camille Guérin
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• Chromatographic analysis
• Depend on the analysis of fatty acids by Gas
  chromatography or HPLC.
• Molecular Methods
• These methods are based on the detection of a
  specific gene(s) of the TB. Of these methods
• ? DNA probe method (Nucleic acid hybridization).
• Nucleic acid probes are commercially available.
  The detection is done within 2-3 hours.

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• ? PCR method.
• It is a rapid and sensitive method and does not
  require the presence of the organism but only a
  specific DNA fragments to be present.
• The limitations of these two methods are
• The cost, technical expertise.
• The unavailability of the organism to do
  antibiotic sensitivity testing.

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• Prevention and Control
• B.C.G. vaccine it is a living attenuated
  vaccine derived from M. bovis. Attenuation is
  obtained by repeated subcultring (about 250
  times) on unsuitable medium containing
  glycerol, potato and bile.
• Hygienic measures, etc.
• Eradication of infected animals (tuberculin ve)
  by slaughtering.
• Good nutrition and effective pasteurization of
  milk.

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• Treatment
• Because of drug resistance, antibiotic
  sensitivity test should be
  performed.
• Resistance by mutation.

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Atypical or Anonymous
MycobacteriumMycobacterium other than
tuberculosis (MOTT)
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• General Features
• Acid fast bacilli differ from the typical TB in
  being non pathogenic to guinea pig.
• They can be treated with antibiotics and they are
  resistant to anti-tuberculous drugs.
• They are widely distributed in the environment.
• They form smooth and pigmented colonies.
• They are classified according their culture
  characteristics into

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Atypical or Anonymous
Mycobacterium
Slow Growing
Rapid Growing
Photochromogenic
Scotochromogenic
Non Chromogenic
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• ?Slow growing
• A. Photochromogenic
• Produce yellow pigment when the growth is being
  exposed to light.
• Example M. kansasii, M. marinum.
• B. Scotochromogenic
• Produce orange color chiefly in the dark.
• Example M. scrofulaceum.

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• C. Non Chromogenic
• Produce no pigments.
• Example M. avium -intracellular complexes.

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• ? Rapid growing
• Grow within few days and they can grow in
  ordinary media.
• They can produce several types if infection even
  mimics TB in symptoms.
• Examples
• M. fortuitum -chelonei complex.
• M. smegmatis is a rapidly growing
  mycobacterium that is not associated with human
  disease. It is part of the normal flora of
  smegma, the material that collects under the
  foreskin of the penis.

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Typical mycobacterium Atypical mycobacterium
Culture Characteristics - Produce dry, warty and buff colonies (human type) or soft white colonies (bovine type). Require enriched media. Give smooth and pigmented colonies. Can grow on ordinary media.
Catalase test -ve Strongly ve
Niacin test ve (human type) -ve (bovine type) ve
Pathogenicity to guinea pigs Pathogenic Non pathogenic
Susceptibility to anti-tuberculous normally susceptible Not
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Mycobacterium leprae (The causative agent
of leprosy)
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• Morphology
• They are acid and alcohol fast bacilli when
  stained with modified Ziehl- Neelsen stain (5
  H2 SO4).
• They may appear beaded but coarser than TB.
• They arrange in masses or groups and mostly
  intracellular.
• Non spore forming.
• Non motile.

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• Culture
• They cannot grow on any artificial media or cell
  culture.
• It can be grown in the mouse footpad or in the
  armadillo.
• Humans are the natural host.
• The optimum temperature for growth is 30 C. It
  is lower than body temperature it thus grows
  preferentially in the skin and superficial
  nerves.

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• Epidemiology
• Transmission Infection is acquired by prolonged
  contact with patients with lepromatous leprosy,
  who discharge M. leprae in large numbers in nasal
  secretions and from skin lesions.
• The incubation period is extremely long, lasting
  from several months to 20 years.
• The disease occurs worldwide, with most cases in
  tropical regions of Asia and Africa.

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• Pathogenicity
• Leprosy in man occurs in three clinical forms
• ?The nodular or lepromatous type
• - In which the organisms produce nodules and form
  granulation in skin, mucous membrane and internal
  organs.
• ?Tuberculoid type.
• In this case the nerve endings are usually
  affected with paralysis or loss of sensation of
  the affected area.
• ?Mixed type.

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• Laboratory Diagnosis
• ? The specimen
• Collected from any ulcerated nodules of the skin
  or from the nasal secretion as well as by gentile
  scraping of the nasal septum.
• ? Direct Examination
• Film stained with modified Ziehl Neelsen stain,
  the presence of acid and alcohol fast bacilli,
  usually in groups, intracellular is diagnostic.

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• ? Indirect allergic test or Lepromin test
• It is similar to tuberculin test however, the
  results are unreliable.
• Immunization
• BCG can also be given to protect against leprosy
  especially for individuals between 10-
  29 years old.
• Treatment
• Dapsone in combination with clofazimine and
  rifampin.

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• Prevention and Control
• No vaccine is available.
• Isolation of cases and their treatment for
  2 years or until the disappearance of
  symptoms.
• Chemoprophylaxis with dapsone of exposed
  children.

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Actinomyces and Nocardia
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• General Features
• They are true bacteria (related to corynebacteria
  and mycobacteria), but they form long branching
  filaments that resemble the hyphae of fungi.
• They are microaerophilic or anaerobic on primary
  isolation.
• Nocardia are aerobic organism.

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Actinomyces Nocardia
Staining characters Gram-positive Weakly Gram-positive and acid-fast
Oxygen requirement Strict or facultative anaerobes Strict Aerobes
Epidemiology Part of normal flora (oropharynx, gastrointestinal tract) Prevalent in soil occasionally found in sputum of normal individuals.
Clinical disease Produce abscesses with yellow granules (sulfur granules) Cause subcutaneous and pulmonary infection
Treatment Penicillin G Sulfonamides
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Actinomyces israelii
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• General Features
• Gram-positive bacteria which grow in filaments
  that readily break up into rods and may show
  branching.
• Non-motile.
• Non-spore-forming.
• Non-acid fast.

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• In tissue, colonies develop to show the
  diagnostic yellow "sulfur granules", which is
  visible by naked eye and is found in pus
  discharge through draining sinuses
• Culture characteristics
• A washed, crushed sulfur granule should be used
  for culture.
• Grow on blood or serum glucose agar
  anaerobically. Incubation at 37 C for at least 7
  days.

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• The growth is enhanced by 5 CO2.
• Colonies are small, cream, adherent and nodular.

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• Pathogenesis and Clinical disease
• Actinomyces is endogenous in origin and results
  in a chronic granulomatous infection with abscess
  formation.
• Profuse pus discharges by draining through
  sinuses.
• Infection probably starts after local trauma,
  e.g. the extraction of carious teeth,
  appendectomy.
• Intra-utrine contraceptive devices are often
  colonized with Actino israelii.

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• The significance of this uncertain but, perhaps
  in association with other organisms, the organism
  may cause low-grade intra-utrine infection.
• Treatment
• Penicillin, licomycin or tetracyclines.

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• Nocardia asteroids
• Pathogenesis and Clinical disease
• Generally cause granulomatous suppurative
  infections.
• It affects lungs with secondary brain abscess.
• It is opportunistic pathogen and affects usually
  immunocompromized patients.

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• Nocardia madurae and N. brasiliensis
• Affect subcutaneous tissues madura foot" or
  mycetoma which is a tropical form of nocardiosis
  and affects foot and produces chronic
  discharging sinuses.
• Treatment
• Surgical treatment.
• Trimethoprim-sulfamethoxazole.
•  

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