Mycobacterium tuberculosis

Mycobacterium tuberculosis
Dr. Pendru Raghunath Reddy

Mycobacteia are slender rods that sometimes show branching,
filamentous forms resembling fungal mycelium

Classification
The genus Mycobacterium contains three groups

1.Obligate parasites

2.Opportinistic pathogens
3.Saprophytes

Obligate parasites
Mycobacterium tuberculosis complex
Contains M. tuberculosis, M. bovis, M. africanum, M. microti,
M. canetti, M. caprae and M. pinnipedii

Mycobacterium leprae

Opportunistic pathogens
Non-tuberculous mycobacteria (NTM)
This group contains mixed group of isolates from diverse
sources: birds, cold-blooded and warm-blooded animals,
from skin ulcers, and from soil, water and other
environmental sources

They are opportunistic pathogens and can cause many types
of disease

Mycobacterium tuberculosis
• long, slender, straight or curved,
about (3 x 0.3 µm in size)
• Aerobe
• Acid fast bacilli
• Intracellular
• Mycolic acid, waxes & lipids
in cell wall
• Slow growing
(Doubling time: 15 – 20 hours)

The Nobel Prize in Physiology or Medicine
1905

In 1882 while working in Berlin
he discovered the tuberculosis bacteria
and the means of culturing it

Pathogenesis
Source of infection
Open case of pulmonary tuberculosis

Mode of infection
Direct inhalation of aerosolised bacilli contained in the
droplet nuclei of expectorated sputum
Infection also occurs infrequently by ingestion for example,
through infected milk, and rarely by inoculation

Transmission of M. tuberculosis
 Millions of tubercle bacilli in lungs (mainly in
cavities)
 Coughing projects droplet nuclei into the air
that contain tubercle bacilli
• One cough can release 3,000
droplet nuclei
• One sneeze can release tens
of thousands of droplet
nuclei

M. tuberculosis does not spread by:
• Sharing dishes and utensils
• Using towels and linens
• Handling food
• Sharing cell phones
• Touching computer keyboard

The initial infection with M. tuberculosis is referred to as a
primary infection
Subsequent disease in a previously sensitized person, either
from an exogenous source or by reactivation of a primary
infection is known as postprimary tuberculosis

Both forms exhibit quite different pathological features

Primary tuberculosis
It is the initial infection by tubercle bacilli in a host

The site of the initial infection is usually the lung
These bacilli engulfed by alveolar macrophages, multiply
and give rise to a subpleural focus of tuberculous
pneumonia
Which is commonly located in the lower lobe or lower part
of the upper lobe to form the initial lesion or Ghon focus
Some bacilli are carried to the hilar lymphnodes through
macrophages, where additional foci of infection develops

The Ghon focus, together with the enlarged hilar
lymphnodes, form the primary complex
M. tuberculosis multiply within the alveolar macrophages
Th-1 cells produce cytokines to activate these macrophages
Activated macrophages effectively destroy most of the
tubercle bacilli
However, some bacilli escape the macrophage- mediated
destruction and induce the hypersensitivity reaction
A hard tubercle or granuloma is formed due to the
hypersensitivity reaction

When fully developed, tubercle/granuloma consists of 3 zones
1. A central area of large, multinucleated giant cells containing
tubercle bacilli
2. A mid zone of pale epitheloid cells, often arranged radially
3. A peripheral zone of fibroblasts, lymphocytes and
monocytes

 Later, peripheral fibrous tissue develops, and the central
area undergoes caseation necrosis
 A caseous tubercle may break into a bronchus, empty its
contents there, and form a cavity
 It may subsequently heal by fibrosis or calcification

Tubercle or granuloma formation in tuberculosis

Postprimary (secondary) tuberculosis
It is due to reactivation of latent infection or exogenous
reinfection and differs from the primary type in many respects

It is characterised by chronic tissue lesions, the formation of
tubercles, caseation and fibrosis
Regional lymphnodes are only slightly involved, and they
do not caseate
Postprimary tuberculosis always begins at the apex of the lung,
where the oxygen tension is highest
The necrotic materials break out into the airways, leading to
expectoration of bacteria-laden sputum, which is the main source
of infection to contacts

Differences beween primary and postprimary
tuberculosis
Characteristics
Site
Local lesion
Cavity formation
Lymphatic
involvement
Infectivity*
Local spread
*Pulmonary cases

Primary

Postprimary

Any part of lung Apical region
Small
Large
Rare
Frequent
Yes
Minimal
Uncommon
Uncommon

Usual
Frequent

Immunology
Tubercle bacilli do not contain or secrete a toxin

The exact basis of their virulence is not understood, but
seems to be related to their ability to survive and multiply
in macrophages
Humoral immunity appears to be irrelevant
The only specific immune mechanism effective is the CMI

The key cell is the activated CD4+ helper T cell which can
develop along two different paths: The Th1 and Th2 cells
Th1 dependent cytokines activate macrophages, resulting
in protective immunity and containment of the infection
Th2 cytokines induce delayed type hypersensitivity (DTH),
tissue destruction and progressive disease

Koch’s phenomenon
Koch’s phenomenon is a combination of hypersensitivity
and immunity
It is the response of a tuberculous animal to reinfection

When a healthy guinea pig is inoculated subcutaneously
with virulent tubercle bacilli, the puncture site heals quickly

After 10-14 days, a nodule appears at the site of injection
which ulcerates and the ulcer persists till the animal dies of
progressive tuberculosis

If on the other hand, virulent tubercle bacilli are injected in a
guinea pig, which had received a prior injection of tubercle bacilli
4-6 weeks earlier, an indurated lesion appears at the site of injection
in a day or two which undergoes necrosis to form a shallow ulcer

This ulcer heals rapidly without involvement of the regional
lymphnodes or tissues. This is called Koch’s phenomenon

Koch’s phenomenon has got three components
1. A local reaction of induration and necrosis

2. A focal response in which there occurs acute congestion
and even hemorrhage around the tuberculous foci in tissues
3. A systemic response of fever that may sometimes be fatal

Laboratory diagnosis
Specimen collection
Type of lesion

Specimen

Pulmonary tuberculosis

Sputum
Laryngeal swabs or
bronchial washings
Gastric lavage

Renal tuberculosis

Urine

Tuberculous meningitis

CSF

 Early morning sputum samples should be collected for 3
consecutive days in a sterile container
 In case of renal tuberculosis, 3-6 morning urine samples
should be collected

Concentration of specimens
Concentration of a specimen is done to achieve;
1. Homogenisation of the specimen

2. Decontamination i.e. to kill commensal bacteria

3. Concentrate the bacilli in the specimen without inactivation
 The concentrate is used for smear preparation, cultutre and
animal inoculation
 Petroff’s method is used to concentrate sputum specimens

Direct Microscopy
 Ziehl-Neelsen staining
(hot staining method)
 Kinyoun’s method
(cold staining method)
 Acid fast bacilli resist decolourisation with acid and alcohol
once they have been stained with carbolfuchsin
 AFB appear as pink, long, slender bacilli with beaded
appearance

Fluorescent staining by Auramine O or
auramine rhodamine

 Mycobacterium spp. will
fluoresce yellow against
dark background under
fluorescent microscope

Diagnosis of pulmonary tuberculosis
under RNTCP

DOTS: Directly observed treatment short-course

Culture
 Concentrated specimen is inoculated
on Lowenstein – Jensen’s medium and
incubated at 370C for 2 – 8 weeks

M. tuberculosis

 Colonies appear as buff coloured, dry,
irregular colonies with wrinkled surface
and not easily emulsifiable
(Buff, rough and tough colonies)

 Colonies are creamy white to yellow colour
with smooth surface and easily emulsifiable
M. bovis

Differentiating features of M. tuberculosis and
M. bovis

Biochemical reactions
Niacin test
 M. tuberculosis lacks the enzyme that converts Niacin to
Niacin ribonucleotide due to this large amount of Niacin
accumulates in the culture medium
 Niacin is detected by addition of
10% cyanogen bromide and
4% aniline in 96% ethanol
 Positive reaction – canary yellow

 M. tuberculosis – Positive
 M. bovis - Negative

Nitrate reduction test
 M. tuberculosis produce an enzyme nitro reductase which
reduces nitrate to nitrite
 This detected by colorimetric reaction
by addition of sulphanilamide
and n-naphthyl- ethylene diamine
dihydrochloride
 Positive reaction – pink or red colour

 M. tuberculosis – Positive
 M. bovis - Negative

M. tuberculosis is resistant to TCH (Thiophene - 2
- carboxylic acid hydrazide); hence, growth occurs
Growth in presence of TCH
M. bovis is susceptible; therefore,
does not grow
M. tuberculosis

M. bovis

Rapid culture methods
1. BACTEC
2. Mycobacterial growth indicator tube (MGIT)

3. Bac T/ Alert 3D system

BACTEC system

 Average time to detect Mycobacterium
growth is 8 days
 Radio metric method
 Detects the presence of Mycobacteria
based on their metabolism rather than
visible growth

 0.5 ml of processed sample is added to 4 ml of Middlebrook
7H12 broth containing C14 radio labelled palmitic acid
 Mycobacteria metabolises C14 radio labelled palmitic acid and
release radio actively labeled 14CO2
 BACTEC 460 instrument measures
14CO and reports in terms of growth
2
index (GI)
 A growth index of 10 or more is
considered positive

 More sensitive than traditional method
 Problem of disposal of radio active
waste

Animal inoculation
 0.5 ml of concentrated specimen is
inoculated intramuscularly into the
thigh of two healthy guineapigs
 The animals are weighed prior to
inoculation and thereafter at
weekly intervals

 Tuberculin test is done after 3 – 4 weeks
 Progressive loss of weight and positive tuberculin skin
reaction indicates infection
 One animal is killed after 4 weeks and autopsied, if it shows
no evidence of tuberculosis the other animal is autopsied
after 8 weeks

Autopsy shows
1. Caseous lesion at the site of inoculation
2. Enlarged caseous inguinal lymph nodes

3. Tubercles may be seen in spleen, lungs, liver, or peritoneum
4. Kidneys are not affected

Allergic tests
 Tuberculosis infection leads to the development of delayed
hypersensitivity to M. tuberculosis antigen, which can be
detected by Mantoux test

Mantoux test (tuberculin test)

 0.5 ml of PPD containing 5 TU is
injected intradermally on flexor
aspect of fore arm

 Site is examined after 48 – 72 hrs

 Induration of 10 mm or more is
considered positive

 Positive tuberculin test indicates
hypersensitivity to tuberculoprotein
denoting infection with tuercule bacilli
or BCG immunisation, recent or past
with or without clinical disease

Uses
1. To diagnose active infection in infants and young children

2. To measure the prevalence of infection in community

3. Indication of successful BCG vaccination

Detection of antibodies
 Various methods such as enzyme linked immunosorbent assay
(ELISA), radio immunoassay (RIA), latex agglutination assay
have been employed for detection of antibodies in
patient serum
 However, diagnostic utility of these methods is doubtful
 WHO has recommended that these tests should not be used
for diagnosis of active tuberculosis

Quantiferon-Gold
 Is an in vitro assay that measures the cell mediated immune
-response in the infected individuals through the levels of
interferon gamma (IFN-γ) released by the sensitised
T- lymphocytes after stimulation by M. tuberculosis antigens

Molecular methods
1. Polymerase chain reaction (PCR)
2. LAMP
3. Ligase chain reaction

PCR
 Rapid method to detect M. tuberculosis directly in clinical
samples based on DNA amplification
 IS6110 sequence is generally targeted for detection
M. tuberculosis complex

Prophylaxis
General measures
Adequate nutrition, good housing and health education are as
important as specific antibacterial measures

Immunoprophylaxis
The BCG (Bacille Calmette-Guerin) vaccine (0.1 ml), administered
soon after birth by intradermal Injection failing which it may be
given at any time during the first year of life
This is a strain of M. bovis attenuated by 239 serial subcultures
in a glycerine-bile-potato medium over a period of 13 years

Bacille Calmette-Guérin = BCG!

Albert Calmette

Camille Guérin

Chemoprophylaxis
This is the administration of antituberculous drugs
(usually only isoniazid)

1. To persons with latent tuberculosis (asymptomatic tuberculin
positive)
2. To persons with a high risk of developing active tuberculosis
3. To the infant whose mother with active tuberculosis
4. To the children living with a case of active tuberculosis in the
house
 Isoniazid 5 mg/kg daily for 6 – 12 months is the usual course

Mycobacterium tuberculosis

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Editor's Notes