Mycobacterium tuberculosis Mycobacterium tuberculosis (M. tuberculosis) is a pathogenic bacterium that causes tuberculosis (TB), a serious infectious disease primarily affecting the lungs. Characteristics 1. *Acid-fast*: M. tuberculosis is an acid-fast bacillus, meaning it retains certain stains even when treated with acid. 2. *Slow-growing*: M. tuberculosis has a slow growth rate, which can make diagnosis and treatment challenging. 3. *Ziehl-Neelsen stain*: M. tuberculosis can be identified using the Ziehl-Neelsen stain, a special staining technique. Transmission 1. *Airborne transmission*: M. tuberculosis is primarily spread through the air when an infected person coughs, sneezes, or talks. 2. *Close contact*: People in close contact with an infected individual are at higher risk of transmission. Pathogenesis 1. *Inhalation*: M. tuberculosis is inhaled into the lungs, where it infects alveolar macrophages. 2. *Intracellular survival*: The bacteria can survive inside macrophages, evading the host's immune response. 3. *Granuloma formation*: The immune system responds by forming granulomas, which are clusters of immune cells that attempt to contain the infection. Clinical manifestations 1. *Pulmonary TB*: Symptoms include cough, chest pain, coughing up blood, and difficulty breathing. 2. *Extrapulmonary TB*: TB can affect other parts of the body, such as the kidneys, spine, or brain. Diagnosis 1. *Sputum smear microscopy*: Examination of sputum samples for acid-fast bacilli. 2. *Culture*: Growing M. tuberculosis in culture is a definitive diagnostic method. 3. *Molecular tests*: PCR (polymerase chain reaction) and other molecular tests can rapidly detect M. tuberculosis DNA. Treatment 1. *Multidrug therapy*: A combination of antibiotics, typically isoniazid, rifampicin, pyrazinamide, and ethambutol, is used to treat TB. 2. *Long treatment duration*: Treatment typically lasts for 6 months or longer, depending on the case. Challenges 1. *Drug resistance*: The emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) poses significant challenges. 2. *Global burden*: TB remains a major public health problem worldwide, particularly in low- and middle-income countries. Prevention 1. *BCG vaccine*: The Bacillus Calmette-Guérin (BCG) vaccine provides some protection against severe forms of TB, particularly in children. 2. *Infection control*: Measures such as ventilation, masks, and isolation can help prevent transmission. Public Health Measures 1. *Surveillance*: Monitoring and tracking TB cases. 2. *Contact tracing*: Identifying and screening individuals who have been in contact with someone with TB. 3. *Education*: Public awareness campaigns on TB prevention and treatment. M. tuberculosis is a complex pathogen that requires a comprehensive approach to control and eliminate TB. Ongoing research and development of new diagnostics, treatments, and vaccines are crucial to combating this disease.

1. Mycobacterium
tuberculosis

2. Heinrich Herman Robert Koch
(1843 – 1910)
In 1882 while working in Berlin
he discovered the tuberculosis
bacteria and the means of
culturing it
The Nobel Prize in Physiology or Medicine
1905

3. Taxonomy
Order: Actinomycetales
Family: Mycobacteriaceae
Genus: Mycobacterium
Species: M.tuberculosis, M.bovis,M.africanum
M.leprae
M. ulcerans, M. balnei, M. butyricum,
M. phlei, M. stercoris est.

6. Classification
The genus Mycobacterium contains three groups
1. Obligate parasites
2. Opportinistic pathogens
3. Saprophytes

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

8. M.tuberculosis complex
• M.tuberculosis complex refers to a closely related group of
species, which cause tuberculosis, a chronic granulomatous
disease, affecting man and many other mammals:
• M.tuberculosis, which cause most human tuberculosis;
• M.bovis, which is the principal cause of tuberculosis in
cattle
and many other mammals ;
• M.africanum, which appears to be intermediate between
the human and bovine types. It causes human tuberculosis
and found mainly in equatorial Africa.
• M. microti, M. pinnipedii and M. caprae can cause
tuberculosis in rare cases.

9. 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

10. Atypical mycobacteria Runyon Groups

11. Morphology
• M. tuberculosis is a slender, straight or slightly curved rod
with rounded ends , about 3 µm × 0.3 µm, in pairs or as
small clumps.
• The bacilli are nonmotile
• Nonsporing
• Noncapsulated and acid-fast.
• They are gram-positive, but are difficult to stain
• Acid-fast bacilli (When stained with carbol fuchsin by the
Ziehl-Neelsen method Tubercle bacilli stain bright red,
while the tissue cells and other organisms are stained
blue)

12. Ziehl-Neelsen stain

13. Acid fastness has been ascribed variously to the presence in the bacillus of an
unsaponifiable wax (mycoloic acid) or to a semipermeable membrane around the cell.

17. The cell wall is composed of mycolic acids, complex waxes,
and unique glycolipids.
The lipid components comprise 60% of cell wallweight
(lipids, glycolipids, peptidoglycolipids and
lipoarabinomannan).
Important wall components are trehalosedimycolate (so-
called cord factor, as it is thought to induce growth in
serpentine cords on artificial medium) and mycobacterial
sulfolipids, which may play a role in virulence.
Another unique constituent which may contribute to
pathogenesis is lipoarabinomannan (LAM).

18. Cord-factor. Ziehl-Neelsen stain.
(serpentine arrangement of virulent strains)

19. Cord factor is component of the cell wall of
tuberculosis mycobacteria trehalose
dimycolate, which possesses adhesive,
antiphagocytic and toxigenic properties
(inhibits respiratory chain in mitochondria
of eukaryotic cells).
The virulent mycobacteria form cords
during cultivation on slides during 3-7 days
due this component of cell wall. The
method is called microculture method
(Prayth’s method).

20. L-forms.
One of the important features of MBT is its ability
to produce L-forms The L-forms are characterized
by reduced level of metabolism and weak virulence.
Remaining viable L-forms can survive for a long
time and produce anti-tubercular immunity. The L-
form differs from usual MBT by the expressed
functional and morphological alterations.
It has been discovered, that the transformation of
MBT into the L-forms accelerated under long anti-
bacterial therapy and under other factors, which
inhibit the MBT growth, duplication and cell
membrane formation

21. Cultural Characteristics
M. tuberculosis is an obligate aerobe (M. bovis is
microaerophilic on primary isolation, becoming aerobic
on subculture).
The optimal growth temperature of tubercule bacilli is
35 to 37°C but they fail to grow at 25°C
or 41°C. Optimum pH is 6.4 to7.0.
The solid media contain egg (Lowenstein Jensen,
Petragnini, Dorset), blood (Tarshis), serum (Loeffler) or
potato (Pawlowsky).
Colonies appear in about two weeks and may
sometimes take up to eight weeks

22. Among the several liquid media described,
Dubos’, Middlebrook’s, Proskauer and Beck’s,
Sula’s and Sauton’s media are the more
common.
Dubos’ medium (Twin-80) and Middlebrook 7H9
are two commonly used liquid media. Liquid
media are not generally employed for routine
cultivation but are used for sensitivity testing,
chemical analyzes and preparation of antigens
and vaccines

23. Lowenstein-Jensen medium ( glycerated egg-based medium)
(used for growing Mycobacterium tuberculosis Malachite green, prevent the
growth of the majority of contaminants surviving decontamination of the
specimen while encouraging the growth of Mycobacteria)

24. • M. tuberculosis on solid media forms dry, rough,
raised, irregular colonies with a wrinkled surface.
They are creamy white, becoming yellowish or buff
colored on further incubation.
• Growth on LJ medium in about 2 weeks, although on
primary isolation from clinical material colonies may
take up to 8 weeks to appear.
• Mycobacterium tuberculosis has a luxuriant growth
on LJ medium.
• Mycobacterium bovis colonies - smooth, moist, white
and which grows poorly on LJ glycerol medium. The
growth of M. bovis is much better on LJ pyruvate
medium (media containing sodium pyruvate in place
of glycerol).

27. Atypical mycobacteria Runyon Groups

30. Virulence factors of M.tuberculosis
1. Cord-factor (a toxic 6,6'-diester of trehalose
containing mycolic acids) disrupts respiration in
mitochondria of eukaryotic cells and possesses
antiphagocytic activity, it has also adhesive,
colonization and antiphagocytic properties
2. Wax D (peptidoglycolipid, containing mycolic acids)
is antiphagocytic factor. Wax D forms the additional
layer, alter the metabolism of the host cell
3. Wax С (dimecosarate ftiocerole) is antiphagocytic
factor and suppress secretion of lysosomal enzymes;

31. 4. Sulpholipids inhibit action of lysosomal enzymes
(antiphagocytic factor);
5. Phthionic acids + Wax D + tuberculoproteins
(Tuberculin) is the complex of protein substances mand
contains also the high molecular polysaccharides, which
release during disruption of Mycobacteria (causes cell-
mediated hypersensitivity).
6. Mycosides and lipoarabinomannan - determine the
attachment of microbes to macrophage cell receptors
and initiate “non-aggressive phagocytosis” of
mycobacteria

32. • !!!!! Mycobacteria are able to survive and multiply
in macrophages
• !!!!Tubercle bacilli do not produce exotoxin or
endotoxin

33. 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

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

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

36. ➢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

38. 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

39. ❑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

40. 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

42. nterferon-γ release assays

44. Tubercle or granuloma formation in tuberculosis

45. 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

46. Characteristics Primary Postprimary
Site Any part of lung Apical region
Local lesion Small Large
Cavity formation Rare Frequent
Lymphatic
involvement
Yes Minimal
Infectivity* Uncommon Usual
Local spread Uncommon Frequent
*Pulmonary cases
Differences beween primary and postprimary
tuberculosis

48. Laboratory diagnosis
❑ 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
Type of lesion Specimen
Pulmonary tuberculosis Sputum
Laryngeal swabs or
bronchial washings
Gastric lavage
Renal tuberculosis Urine
Tuberculous meningitis CSF

49. Concentration of specimens
Concentration of a specimen is done to achieve;
1. Homogenisation of the specimen
2. Concentrate the bacilli in the specimen without inactivation
➢ The concentrate is used for smear preparation, cultutre and
animal inoculation
➢ Petroff’s method (Prayth’s method) is used to concentrate
sputum specimens

50. Diagnostic Methods

53. Direct Methods

54. 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

56. Fluorescent staining by Auramine O or
auramine rhodamine
❑Mycobacterium spp. will
fluoresce yellow against
dark background under
fluorescent microscope

57. Culture
❑ Concentrated specimen is inoculated
on Lowenstein – Jensen’s medium and
incubated at 370C for 2 – 8 weeks
❑ 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
M. tuberculosis

58. Distinguishing features of Mycobacterium tuberculosis and M. bovis

59. Biochemical Reactions
1. Niacin Test
2. Nitrate reduction test
3. Catalase Activity
4. Tween 80 Hydrolysis
5. Arylsulfatase Test
6. Urea Hydrolysis test

61. 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

62. 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

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

64. 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

65. ❑ 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
14CO2 and reports in terms of growth
index (GI)
❑ A growth index of 10 or more is
considered positive
❑ More sensitive than traditional method
❑ Problem of disposal of radio active
waste

66. 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

67. 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

68. 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

69. ✓ 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

70. 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

71. Quantiferon-Gold
(Interferon-γ release assays)
✓ 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

72. 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

75. 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

76. Bacille Calmette-Guérin = BCG!
Albert Calmette Camille Guérin

77. 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