MYCOBACTERIA
Dr. Sudheer Kher
KEYWORDS
Acid Fast
 Ziehl-Neelsen Stain
 Mycolic acids
 Tuberculosis (Koch’s
disease)




M. tuberculosis
M. bovis
M. leprae
Tubercle
 Lowenstein Jensen
medium

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



PPD
Tuberculin
BCG
Polymerase chain
reaction
Runyon groups
Classification of Mycobacteria
1.
Tubercle bacilli
a)
b)
c)
d)
e)
2.
Lepra bacilli
a)
b)
3.
Human – MTB
Bovine – M. bovis
Murine – M. microti
Avian – M. avium
Cold blooded – M.
marinum
Human – M. leprae
Rat – M. leprae murium
Mycobacteria causing
skin ulcers
a)
b)
M. ulcerans
M. belnei
4.
Atypical Mycobacteria
(Runyon Groups)
a)
b)
c)
d)
5.
Photochromogens
Scotochromogens
Nonphotochromogens
Rapid growers
Johne’s bacillus
M. paratuberculosis
6.
Saprophytic
mycobacteria
a)
b)
c)
d)
e)
M. butyricum
M. phlei
M. stercoralis
M. smegmatis
Others
Tuberculosis
(TB, Consumption, Koch’s Disease)
• M. tuberculosis
• major human disease
– healthy people
• problems
– association with AIDS
– multiple drug-resistance
–Chronic disease
–Prolonged treatment
General characters of the genus
Slender rods
 Resist staining but
once stained, resist
decolorization by
dilute mineral acids;
hence called ACID
FAST BACILLI (AFB)

Aerobic, Non-motile,
Non-sporing, Noncapsulated.
 Growth generally slow
 Genus includes

– Obligate parasites
– Opportunist pathogens
– Saprophytes
Mycobacterium tuberculosis
One of the most serious infectious diseases in
the developing world
 One third of world’s population infected with M.




tuberculosis
Thirty million people have active disease
Nine million new cases occur
Three million people die of the disease, each
year.
Mycobacterium tuberculosis (MTB)

Morphology –
– Ziehl – Neelsen stain – Once stained by Carbol
fuchsin, resist decolorization by 20% Sulphuric acide
and absolute alcohol. Acid & Alcohol Fast (AFB)
– Fluorescent dyes like Auramine O or Rhodamine also
stain and the decolorization is resisted.
– Reason for Acid & Alcohol fastness –
 Presence of unsaponifiable wax Mycolic acid
 Semi permeable membrane around the cell
 Property of cell wall and related to integrity of the cell wall
– Staining may be uniform or granular
MTB : Cultural characters
 Grow
slowly.
Generation time
14-15 hrs
 Colonies appear
after 2 weeks or
at 6-8 weeks
 MTB - Obligate
aerobe
MTB grows more
luxuriantly (eugonic)
than M. bovis
(dysgonic).
 Addition of 0.5%
Glycerol supports
growth of human
strains. No effect or
inhibitory effect on
bovine strains.

MTB : Culture media

Solid media –
– Egg containing
 Lowenstein-Jensen
Medium
 Petragnini medium
 Dorset
– Blood containing –
 Tarshi’s
– Serum containing –
 Loeffler’s serum slope
– Potato containing –
 Pawlowsky’s

Liquid media –
–
–
–
–
–
Dubos’
Middlebrooks
Proskauer & Beck’s
Sula’s
Sauton’s
Resistance
Not specifically resistant to heat. 60 C x 20 min
destroys.
 In sputum can survive 20-30 hrs
 Relatively resistant to disinfectants. Survives
exposure to

–
–
–
–
–
5 % Phenol
15 % Sulphuric acid
3 % Nitric acid
5 % Oxalic acid
4 % NaOH
Biochemical reactions

Niacin test – Human MTB produces niacin when
grown in egg medium.

Aryl Sulphatase test – Enzyme Aryl sulphatase
formed by only atypical mycobacteria.
Biochemical reactions
Neutral red test – Virulent strains of tubercle bacilli
bind neutral red in alkaline solution while avirulent
strains can not.
 Catalase–paroxidase test – Most atypical
mycobacteria are strongly catalase positive while
MTB is only weakly positive. MTB is strongly
peroxidase positive while atypical mycobacteria are
negative.
 Nitrate reduction test – Positive in MTB and
negative in M. bovis

Mycobacterium tuberculosis (MTB)

Morphology –
– Straight or slightly
curved rods
Modes of infection
1- Droplet infection
Person to person by inhalation aerosols
Mycobacterium tuberculosis (Pulmonary tuberculosis)
2- Ingestion of milk
Infected cattle
Mycobacterium bovis (Intestinal tuberculosis)
3- Contamination of abrasion
Laboratory workers
(Skin infection)
Pathogenesis of
tuberculosis
• infects lung
• distributed within macrophages
• facultative intracellular pathogen
– inhibits phagosome-lysosome fusion
– resists lysosomal enzymes
Other minor pathogenesis factors
tuberculosis
• mycobactin
– siderophore
• cord factor
– damages mitochondria
Cell-mediated immunity tuberculosis
•
infiltration
– macrophages
– lymphocytes
granulomas
• tubercles
•
Classical tubercular lesion – Granuloma with typical
Langhan’s giant cells, epithelioid cells, lymphocytes
and fibrosis.
There are multiple light areas (opacities) of varying size that
run together (coalesce). Arrows indicate the location of
cavities within these light areas. The appearance is typical
for chronic pulmonary tuberculosis.
Tuberculosis
Clinical picture :
*
*
*
*
*
Low grade fever
Weight loss
Night sweats
Fatigue
Cough & haemoptysis
Laboratory diagnosis
M. tuberculosis
Acid fast bacteria in sputum
 Culture on L J media
 Biochemical identification
 Antibiotic sensitivity test
 Tuberculin test
 PCR

Lowenstein Jensen Medium –
Selective. Always in screw capped bottle. Bluish Green.
Contains – Egg protein – Solidifying agent
Mineral salts – Mg sulphate, Mg citrate
Asparagine
Malachite Green – Selective agent
Sterilized by - Inspissation
Laboratory diagnosis -
tuberculosis
•
skin testing
– delayed hypersensitivity
– tuberculin
– protein purified derivative,
PPD
•
X-ray
Tuberculin Test (Mantoux test)

Delayed hypersensitivity skin
test to assay:
cell mediated immunity to
tubercle bacillius
Material: A purified protein
derivative (PPD)
Dose
: 0.1 ml of (PPD) is
injected intradermal
Reading : Positive test is
defined as
- Induration equal or
greater than 10 mm
- Develop 48-72 hours
after injection
Positive skin test tuberculosis
indicates exposure to
organism
• does not indicate active
disease
•
Tuberculin Test
Interpretation:
* A positive test indicates previous exposure and carriage of T.B.
* A negative tuberculin test excludes infection in suspected persons
* Tuberculin positive persons may develop reactivation type of T.B.
* Tuberculin negative persons are at risk of gaining new infection
* False positive reactions are mainly due to:
- Infection with nontuberculous mycobacteria
* False negative reactions may be due to:
- Sever tuberculosis infection (Miliary T.B.)
- Hodgkin’s disease
- Corticosteroid therapy
- Malnutrition
- AIDS
* Children below 5 years of age with no exposure history:
- Positive test must be regarded suspicious
Laboratory Diagnosis
Demonstration of bacilli
 Culture & isolation or Animal inoculation
 Demonstration of hypersensitivity to
tubercular protein
 Serological tests limited value

Specimen
* According to site of infection :
- Sputum
- Urine
- Gastric lavage
- Blood
- Body fluids
- Tissue biobsy
* Specimens need appropriate processing
Sputum
Liquefaction with N-acetyl-L- cysteine
Decontamination with NaOH
Centrifugation
Laboratory Diagnosis

Pulmonary TB –
– Specimen –
 Sputum – Early morning, if scanty 24 hrs, three consecutive
day samples. Laryngeal swabs or gastric lavage in children.
– Microscopy –See at least 100 field / 10 minutes.
 Grading –
– 1+ -> 3-9 bacilli in entire smear
– 2+ -> 10 or more in entire smear
– 3+ -> 10 or more bacilli seen in most oil immersion
fields
– Culture & AST
– Animal inoculation – Animal of choice Guinea
Pig.
Laboratory Diagnosis

Extra -Pulmonary TB –
– Specimen –
 CSF – in suspected meningitis
 Pleural fluid & other exudates
 2-3 days urine in renal TB
 Biopsy material.
Concentration methods
Purpose – Homogenization &
Concentration in sputum & other
specimens.
 Methods –

Concentration methods

Useful for microscopy, culture & animal
inoculation
– Petroff’s method
 Most widely used
 Equal volumes of Sputum + 4% NaOH incubated at 37C X 20
min.Centrifuge at 3000 rpm X 30 min. Sediment neutralized
by N/10 HCl.
 Can be used for smear, culture, animal inoculation
– Simpler method
 To avoid centrifugation & Neutralization
 Equal volumes of sputum + solution of Cetrimoniom bromide
20 g + NaOH 40 g in one litre. Allow to stand for five
minutes. Inoculate Acid Buffered LJ Medium with swab.
Laboratory diagnosis
M. tuberculosis (culture)
• grows very slowly
– several weeks
– non-pigmented colonies
– niacin production
*differentiates from other mycobacteria
Tuberculosis
•
polymerase chain
amplification
–rapid diagnosis
Pathogenesis
* Inhalation of tubercle bacilli
* They multiply in the alveolar macrophages
* An early tubercle (granuloma) is formed
Pathogenesis
* Lesions, healing or progression of infection
depend upon
1- Dose of infecting mycobacteria
2- Resistance and hypersensitivity of host
* Virulence :
Glycolipids on the outer surface of bacteria
- Enhance granuloma formation
- Inhibit migration of polymorphnuclear
leucocytes
- Help survival of tubercle bacilli inside
macrophages
Pathogenesis
A- Primary infection:
* An exudative lesion :
- spread to regional lymph nodes
- A scar of healing may later calcify (Ghon’
focus)
- Lymph nodes caseate and then calcify
- Bacilli in the lesion slowly die
- Tuberculin test becomes positive
- The person immune & hypersensitive
Pathogenesis
B- Reactivation type :
* Activation of tubercle bacilli due to immunity
* Formation of tubercles that caseate
- fibrosis
- open into a bronchus (open tuberculosis)
* Tubercle bacilli erode a blood vessels
- Infect any organ (Miliary T.B.)
Treatment
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

Chemoprophylaxis – INH for one year
Domicilliary treatment preferred
Drugs –
–
–
–
–
–
–
–
–
–
Rifampicin
Isoniazide
Pyrazinamide
Streptomycin
Ethambutol
Ethionamide
Thiacetazone
Paraminosalicylic acid
Cycloserine
Bactericidal
Bacteriostatic
Treatment
Short term chemotherapy of six months is
sufficient
 Problem area – Development of resistance
by mutant selection

– Solution – Treatment by two to three drug
combination, adequate treatment.
Treatment
Use multiple drug therapy to prevent emergence of
resistant mutants
* Long duration treatment (6-18 months)
* Four drugs are usually started in initial therapy due to:
- Intracellular location of bacilli
- Slow growth rate of bacilli
- Caseous material blocks penetration of drugs
- Some bacilli persist in a metabolically inactive state
* Sputum becomes non-infective 2-3 weeks after starting
therapy
Treatment
Drugs used :
1- First line drugs :
- Isoniazid - Rifampicin - Pyrazinamide
- Ethambutol
- Streptomycin
2- Second line drugs (more toxic and less effective):
- Kanamycin
- capreomycin
- Cycloserin
- ethionamide
- ciprofloxacin
- Ofloxacin
* Noncompliance (failure to complete the course):
Directly observed therapy (DOT)
Health care workers observe the medication
Immuno-prophylaxis
Intradermal injection of live attenuated
vaccine Bacille Calmette-Guerin (BCG).
 The strain causes self limited lesion and
induces hypersensitivity & immunity.
 Coverts tuberculin negative person to
positive reactor.
 Immunity lasts for 10-15 years. Immunity
60-80%

BCG
Given at birth without tuberculin testing
 Protects against TB, the disease runs
milder course in protected, prevents
skeletal, meningeal & miliary forms.
 Also found useful in leprosy, leukaemias
and other malignancies by non-specific
stimulation of RE system.
