Mycobacterium

(Fungus Bacterium)
Classification: (Bergey’s Manual of Systematic Bacteriology)

 Kingdom II: Bacteria

 Phylum VIII: Firmicutes
 Section XXIII: The Actinobacteria
 Class I: Actinobacteria (including: Actinomyces, Corynebacteria, Norcardia, Rhodococcus)
 Order V: Actinomycetals
 Suborder III: Corynebacterinaea
 Family IV: Mycobacteriaceae
 Genus: Mycobacterium (only 1 genus, but >100 species!)

To include a species under this genus, the minimum standard is:

 Acid (alcohol) fastness :- Resists decolourization by acidified alcohol after stained with basic
fuschin)
 Presence of mycolic acid (in cell wall)
 A G+C (Guanine + Cytosine) content of DNA of 61-71 mol %

Genus Mycobacterium

 At least 100 species to date!

 Causative agents for
i. Tuberculosis
ii. Leprosy
iii. Similar diseases in animals
iv. Some saprophytes  opportunists

Tuberculosis

 Disease of great antiquity

 Afflicted Neolithic man (circa 10,000B.C)
 Described in Vedas as “Raja-Yakshma” (~King of diseases)
 Well described by Hippocrates (400 B.C)
 Caused more suffering & death than any other infection
 Was referred to as the “White plague”

History

 Mycobacterium – among first bacteria discovered

 1874: Armauer Hansen – Bacillus Leprae
 1882: Robert Koch – Bacillus Tuberculosis
 1884: Robert Koch – Koch’s Postulates
 Subsequently “bacillus”  Mycobacterium (mould-like pellicle)
  1900’s: Other spp. Discovered (opportunistic, environmental, non-tuberculous)

Mycobacterium: Acid Fast Bacilli

Standard staining methods

 Ziehl-Neelsen (hot staining)

 Kinyoun (cold staining)
 Auramine o fluorescence

Ziehl – Neelsen Stain

 Basic fuchsin + phenol (enhance fuchsin penetration into lipids)

 Wash
 Acid alcohol (decolorizer)
 Wash
 Methylene Blue (Counter stain)

Other Acid Fast Organisms

 Norcardia
 Actinomyces
 Corynebacterium
 Rhodococcus

Culture

 M. Leprae – HAS NOT BEEN GROWN ON CULTURE!

 Others:
- Lowenstein – Jensen (L J) medium
- Middlebrook 7H9 (liquid)
- Middlebrook 7H10 (Agar based)
- Middlebrook 7H11 (Agar based too!)
 Clinical specimens : 7 days – 6 weeks (Sometimes, even up to 12 weeks or more!)
 As a whole, mycobacterium are VERY slow growing

Mycobacterium  Acid Fast Bacilli

 Non-motile
 Non-spore forming
 Weakly Gram (+)
 Aerobic or microaerophilic (M. Tuberculosis is strictly aerobic)
 Straight or slightly curved rod-shaped
 Some coccobacillary
- Filamentous
- Branched forms
 Some spp produced pigments (yellow  orange)
- In the dark (scotochromogens)
 - After exposure to light (photochromogens)
- No-pigmentation (Non-chromogens)
 Some slow growers (>7/7 to form visible colonies)
 Some rapid growers (<7/7)
 1959 – Runyon divided mycobacterium into 4 groups
i. Group I = Photochromogens
ii. Group II = Scotochromogens
iii. Group III = Non-Chromogens
iv. Group IV = Rapid growers
 Groups I-III are slow growers

Mycobacterium:

 Can be isolated from a variety of sources

- Clinical specimens
- Environment (water, soil, dust)
 Can survive for long periods of time

 As pathogens f. M. haemophilum
i. Obligate pathogens g. M. leprae
a. M. tuberculosis h. M. malmoense
b. M. bovis i. M. microti
c. M. africanum j. M. paratuberculosis
d. M. ascaticum k. M. shimodei
e. M. farcinogens l. M. Simiae
m. M. Szulgai

ii. Facultative pathogens

a. M. avium h. M. intracellulare
b. M. chelonae i. M. marinum
c. M. fortuitum j. M. senegalense
d. M. kansasii k. M. scrofulaceum
e. M. ulcerans l. M. xenopi
f. M. terrae m. M. malmoense
g. M. genavense

TUBERCULOSIS
 Chronic granulomatous disease
 Affects humans and many mammals
 At one time – single most important infectious disease of humans ( 1/7th of all deaths
worldwide)
 Presently:
- 1/3 world population infected
 - Cause about 3 million deaths per year (>5% of all deaths)
- Is becoming a reemerging disease because of HIV
 Caused by 4 very closely related spp:
i. M. tuberculosis (Humans)
ii. M. bovis (Cattle)
iii. M. africanum (intermediate of above)
a. Type I: In West Africa (~M. bovis)
b. Type II: In East Africa (~M. Tuberculosis)
iv. M. microti (Vole) – Not in man
 M. canetti (Very rare M. TB c smooth)
 M. bovis variant in good  few TB in vets (M. caprae)

By the way, this is a vole:

Mycobacterium Tuberculosis
 Acid fast bacteria
 Obligate aerobe
 Non-motile
 Non-sporing
 Unencapsulated
 Straight / Slightly curved rod
 3 x 0.3 µm in size
 Grow in several enriched media
LJ – most widely used
(Whole egg, glycerol, asparagine, salt)
+ Malachite green (inhibit contaminants – kills other microorganisms)

M. TB M. bovis M. africanum
Atmospheric Obligate aerobe (on Micro-aerophilic (few Micro-aerophilic
preference surface) mm below surface)
Growth in LJ Heaped up luxuriant Small flat dysgonic Intermediate
eugonic
Reduce nitrate  + - Variable
Nitrite
Niacin production ++ +/- +/-
 Sensitivity to S R S
pyrazinamide
Sensitivity to thiopen-2 R S S
carboxylic acid
hydrazide (TCH)

M. Tuberculosis

 Survive in milk, organic matter and pasture land (no UV!)

 UV very sensitive
 Heat sensitive  Pasteurization
 Susceptible to alcohol, formaldehyde, gluteraldehyde
 Less susceptible to phenolics & hypochloride
 Resistant to acids, alkalis and quarternary NH3 compounds

Pathogenesis

 Virulence – due to invasiveness. It does not produce toxin

 Able to survive in microorganisms
 Immune response = Cell mediated immunity (Type IV HS)
 T helper  protective immunity
 Or  tissue destroying HS reactions  disease

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Primary TB

 Part of entry: Respiratory tract

 Mode: Inhalation of bacilli
 Site: Lungs (usually)

Bacilli  Alveolar microorganisms in lungs multiply  Initial lesion

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
If part of entry – mouth (milk, food)
= Primary complex
 Tonsil
 Cervical lymph node enlarged (Scrofula)
 Intestine (ileocaecal region + mesenteric lymph node)
If part of entry – skin
 Skin + regional lymph nodes = Primary complex (Prospecter’s Wart)

1-2 weeks:

 Usually limit primary infection

Granuloma with central caseation  Quiescent  Fibroblasts  Scar + Calcification

NOT ALL hosts can completely destroy mycobacterium  Dormant (How??)

= immunocompromised  Reactivation (?? Reinfection)  Post-primary TB

STAGES OF PRIMARY TB IN CHILDHOOD

Time from onset Characteristics

3-6 weeks  Primary complex develops
 TB Conversion +
2-6 months  Progressive healing of primary complex
 Possibility of pleural effusion
6-12 months  Possibility of miliary or meningeal TB
1-3 years  Possibility of bone or joint TB
3-5 years  Possibility of gastro-urinary or chronic TB

Just for fun  Balita = Bawah Lima Tahun

POST PRIMARY TB

 Most of those exposed to M. TB

= Primary complex heals completely
 Only evidence (+) = Tuberculin test (+)
 In some, after months/years/decades
= Reactivation or ?? Exogenous infection  Post-primary TB
- Larger local lesions
- Minimal lymphatic involvement
- > “open cases” – infective
- Especially apical region of lungs
 Reactivation
- Spontaneous
- Decrease immune responsiveness
 Process of granuloma formation
= Same except more extensive with large areas of caseation (tuberculomata)
  Proteases & Cytokines (e.g. TNF)
 Process
= Erode wall of bronchus  Liquefied content  Expectorated  Aerobic (open cases) 
Bacilli growth
 In immunocompromised:
- Cavitation rare!
- Lymphatic & hematogenous spread common  Cryptic disseminated TB

Diagnosis

 Clinical
 X-ray
 Tuberculin test
- Old tuberculin
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Purified protein derivative (PPD)
Mantoux test  Intracutaneously
Heaf test  Gun with 6 prongs
Tine test  Dried PPD onto prongs for 1 test (disposable)

PPD activities:-
1:100  100 iu }
1:1000  10 iu } 0.1 ml
1:10000  1 iu }
Standard : 10 iu
iu = international unit

(+) – Induration  φ  <5 mm

 5-10 mm

>10 mm

Interpretation
Endemic areas
Non-endemic areas
Children
Immunocompromised
Lab specimens
= Sputum, bronchial washings, biopsies, gastric aspirates, CSF/Pleural fluid, etc
Microscopy
Culture

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If CSF or Pleural Fluid or Biopsy, no need to decontaminate or centrifuge. Directly
deposit and inoculate.
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DRUG SUSCEPTIBILITY TESTING

 In L.J, containing doubling dilutions of drugs

TREATMENT

Drugs = 3 groups

1. Sterilizing
- Rifampin
- Pyrazinamide
2. Bacteriostatic
- Ethionamide
- Prothionamide
- Thiacetazone
- P-aminosalicylic acid
- Cycloserine
3. Bacteriocidal
- Isoniazid
- Streptomycin
- Ethambutol

Also: Multidrug treatment (MDT)

Other ways to Diagnose TB

 α-PGL (glycolipid)
 PCR
 BCG (Bacillus Calmette-Guerin)
- Protects children for severe TB
- Offers cross protection against Leprosy