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 macrophages
 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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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

Leprosy
(Morbus Hansen [MH])

(Hansen’s disease)

Definition: A chronic mycobacterial disease primarily affecting the peripheral nervous system and
secondarily involving skin and other tissues.

History
● 600 B.C – written records in India
● 150 B.C – written records in China
● 2nd century BC – Egyptian mummies
● From Egypt -> Europe (150 AD: Greece)
● Then -> Americas & other parts of the world
● 1983: WHO -> Estimated total in world – 10.5 million
 ● 1996 (Multiple drug treatment) -> 1.3 million
● Malaysia –Common Orang Asli & Foreign workers
Etiology
Mycobacterium Leprae [unculturable in lab] (Armauer Hansen 1874)

Grown in:

● mouse footpad
● 9 banded armadillos (discovered because of Armauer Hansen’s wife)

Man – only significant reservoir

Transmission:

● via nasal secretions by droplets

● Skin
Pathogenesis
Principal target cell = Schwann cells

Nerve damage

● Anaesthesia
● Muscle paralysis
Skin -> usually first sign/symptom

● Non-specific lesions with pigmentary changes (hypopigmented) – anaesthesia /

hypoaesthesia (Whether the patient still feels pain at the area. Use a needle to prick)
● Often heals spontaneuously
Clinical manifestations depend on immune status of patient

Range TUBERCULOID -> LEPROMATOUS

TUBERCULOID LEPROMATOUS

Hyperactive immune response Anergy

Few localized skin lesions with few bacilli Numerous skin lesions – often confluent
(paucibacillary)

Intense granulomatous response -> Damage Bacilli ++++ (multicacillary)

major nerves (hardening)

Immune response greatly weakened (anergy)

Ear lones – bacilli ++++

● Those with the better immune system will suffer from the tuberculoid form.
● Those with the lepromatous form will have a negative tuberculin test.
● The safest area to wear earings/studs is at the ear lobes. Anywhere else, then it will easily
affect the ear cartilage. It could lead to cauliflower ear. (Pak Nasa’s story)
More WHO criteria:

● Borderline tuberculoid (BT)

● Mid-Borderline (BB)
● Borderline Lepromatous (BL)
Other signs & Symptoms
Eye

● Uveitis

●Corneal infection secondary to eyelid paralysis

●The 2 symptoms above might lead the person to suffer from blindness
Bone resorbtion

● Collapse of nasal bone

● Shortening of fingers & toes (autoamputation)
Neuritis

Orchitis

Immune-Complex Nephritis

Facial skin may become crumpled and look like a lion (Facies leonina)
DIAGNOSIS
History & physical examination

Slit-skin smear and punch biopsy (AFB)

● Ear lobe
● Nasal secretion
● Nasal mucosa
Histology – granuloma + caseation

Skin test

● Lepromin test (only guide)

● Fernandez (early) Rx 1-2/7
● Mutsida (late) Rx 7/7 (Usually negative in Lepromatous Leprosy)
Most cases of Lepromatous Leprosy cases in Malaysia are among the Chinese

Split-skin smear - Acid fast staining

Record # of bacteria/high power

1+ : 1-10 per 100 fields

2+ : 1-10 per 10 fields

3+ : 1-10 per field

4+ : 10-100 per field

5+ : 100-1000 per field

6+ : >1000 per field

= Bacillary Index (BI)

Acid Fast Bacilli:

if viable -> stains strongly and evenly

●
Dead when stains unevenly & weakly
●
Morphological Index (MI):

●Percentage (%) of regularly stained (viable)

●Increase MI – Active disease
●MI decreases with chemotherapy
Other Diagnosis methods:

●α-PGL
●Polymerase Chain Reaction (PCR)
Treatment:

Dapsone (diaminophenyl sulphone –DDS)

● Before 1982 – Standard monotherapy for all forms

DDS – Resistance -> MDT

Add

●Rifampin
●Clofazimine (side effect: Skin discolouration)
Other drugs

● Prothionamide
● Ofloxacin
● Minocycline
WHO

Paucibacillary

●Rifampin
●Dapsone
●(Give these for 6 months)
Multibacillary

●Rifampin
●Dapsone
●Clofazimine
●(Given for 2 years or more)
Reactions:

Lepra/Jopling’s Type I:

● Delayed Type Hypersensitivity

● Vasculitis (Type II HS)
Lepra/Jopling’s Type II

● Erythema Nodosum
● Leprosum
Infections by environmental (Opportunistic) mycobacteria (Pak Nasa said this part is not important)
Runyon’s classification

a. Photochromogens (Group I)
● M. Kansasii
● M. Marinum
● M. Simiae
b. Scotochromogens (Group II)
● M. Scrofulaceum
● M. gordonae
● M. szulgai
c. Non-Chromogens (Group III)
● M. avium
● M. intercellulare
● M. malmoense
● M. xenopi
● M. ulcerans
● M. terrae
d. Rapid Growers (Group IV)
● M. chelonae
Principal types of opportunistic mycobacterial infections in man

Lymphadenopathy

● M. Avium complex
● M. Scrofulaceum
Skin lesions

a.Post-trauma abscesses
● M. Chelonae
● M. Fortuitum
● M. Terrae
b.Swimming Pool Granuloma
● M. Marinum
c.Buruli ulcer
● M. Ulcerans
Pulmonary Disease

● M. Avium complex
● M. Kansasii
● M. Xenopi
● M. Malmoeure
Disseminated Disease

a. AIDS related
● M. Avium complex
● M. Genevense
b. Non-AIDS related
● M. Avium complex
● M. Chelonae