1. MYCOBACTERIUM TUBERCULOSIS
Deepshikha Chhetri
MSc. FSN

2. INTRODUCTION:
 Mycobacteria is an obligate aerobe growing most
successfully in tissues with a high oxygen content.(the upper
lobe of the lung and the kidney).
 Its cell wall contains several complex lipids ( long-chain fatty
acids called mycolic acids).
 It is relatively resistant to acids and alkalis. NaOH is used to
concentrate clinical specimens; it destroys unwanted
bacteria, human cells, and mucus but not the organism.
 They are Facultative intracellular pathogens usually infecting
mononuclear phagocytes (e.g. macrophages).

3. MORPHOLOGY:
 SHAPE - long, slender, straight or slightly curved rod.
 SIZE - 3 x 0.3 µm
 Intracellular.
 Mycolic acid, waxes & lipids are present in
cell wall
 The high lipid content (approximately 60%)
of their cell wall makes mycobacteria acid-fast
and hydrophobic.
 Because the cells are hydrophobic and tend
to clump together, they are impermeable to
the usual stains, e.g. Gram's stain
 Neither gram-positive nor gram-negative.

4. ACID FAST BACILLI:
 Mycobateria are virtually the only bacteria that are
acid-fast because of the presence of mycolic acid and
their lipid-rich cell walls, which are relatively
impermeable to various basic dyes unless the dyes are
combined with phenol.
 Once stained, the cells resist
decolourization with acidified
organic solvents and are
therefore called "acid-fast".
Carbol Fuchsin Stain

5. CULTURE:
 Mycobacteria are aerobes.
 Grow slowly: 14-15 hours
 Optimum temperature: 37degree C.
Do not grow below 25degree C.
 pH between 6.4 to 7.0
 Grow only in specially enriched
media containing egg, asparagine,
potatoes, serum and meat extracts.
 Colonies appear in 2-6 weeks.

6. RESISTANCE:
 Mycobateria can survive in dust for several months in
sputum for 20-30 hours.
 Killed at temperature of 60degree C for 20 minutes and
instantly at 100degree C.
 Sensitive to ultraviolet ray and sunlight.

7. TAXONOMY

8. Species
Growth on
Bacteriologic Media
Preferred Temperature
in Vivo (˚C)
Source or Mode of
Transmission
M. tuberculosis Slow (weeks) 37 Respiratory droplets
M. bovis Slow (weeks) 37
Milk from infected
animals
M. leprae None 32 Prolonged close
contact
Atypical mycobacteria
(ex. M. kansasii)
Slow (weeks) 37 Soil and water
M. marinum Slow (weeks) 32 Water
M. avium-
intracellulare complex
Slow (weeks) 37 Soil and water
M. fortuitum-chelonei
complex
Rapid (days) 37 Soil and water

9. Diseases caused by Mycobacterium species:
 M. leprae - leprosy
 M. avium - lung and skin infections in immunocompromised
hosts
- lymphadenopathy in children
- catheter-related infections
 M. bovis - Primarily infection among the cattle .
It infects Tonsils, Cervical nodes.
Enter through Intestines – infects the ileocecal region.

10. MYCOBACTERIUM TUBERULOSIS
 It causes TUBERCULOSIS which is the most common cause of
death due to bacterial infection worldwide.
 It is closely related to M. bovis. and it is believed to have
evolved from M. bovis after domestication of cattle (8,000-
4,000 BC)
 Archeologists have shown that tuberculosis of the bones
seems to have preceded the disease of the lungs.
 M. tuberculosis is resistant to dehydration and so
survives in dried expectorated sputum; this
property may be important in its transmission by
aerosol.

11. Mycobacterium tuberculosis under Electron Micrograph:

12. Mycobacterium Tuberculosis in Sputum:
Fluorescent Acid Fast Stain

13. HISTORY
 In 1882, microbiologist Robert Koch
discovered the tubercle bacillus, at a time
when one of every seven deaths in Europe
was caused by TB.
 Isolated the mammalian tubercle bacillus
on Heat Coagulated Bovine Serum and
proved its causative role in Tuberculosis.
 He received the Nobel Prize in physiology
and medicine in 1905 for this discovery.

14. TRANSMISSION AND EPIDEMIOLOGY
 M. tuberculosis is transmitted from person to person by respiratory
aerosol, and its initial site of infection is the lung. In the body, it resides
within macrophages.
 In developed countries, tuberculosis is almost exclusively a human
disease, and most tuberculosis is due to reactivation in elderly,
malnourished men.
 In developing countries, M. bovis is found in cow's milk, which, unless
pasteurized, can cause gastrointestinal tuberculosis in humans.
 The risk of infection and disease is highest among socio-economically
disadvantaged people, who have poor housing and poor nutrition.
 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.

15. Pre-Disposing Factors:
 Genetic basis,
 Age
 Stress,
 Nutrition,
 Co existing infections Eg. HIV. HIV association will lead to rapid
spread of tuberculosis. HIV kills CD4+ T Helper cells which normally
inhibit M. tuberculosis.

16. INDIA is the highest TB burden country accounting for more than one-
fifth of the global incidence!!
Indonesia
6%
Nigeria
5%
Other countries
20%
Other 13 HBCs
16% China
14%
South Africa
5%
Bangladesh
4%
Ethiopia
3%
Pakistan
3%
Phillipines
3%
India
21%
Source: WHO Geneva; WHO Report 2014: Global Tuberculosis Control; Surveillance, Planning and Financing
Global annual incidence = 9.4 million
India annual incidence = 1.96 million
INDIA AND TUBERCULOSIS

17.  Estimated incidence
 1.96 million new cases annually
 Estimated mortality
 330,000 deaths due to TB each year
 Over 1000 deaths a day
 2 deaths every 3 minutes
 Someone in the world is newly infected with TB bacilli every
second.
 Overall, one-third of the world's population is currently infected
with the TB bacillus.
 5-10% of people who are infected with TB bacilli (but who are not
infected with HIV) become sick or infectious at some time during
their life.

18. PATHOGENESIS
 M. tuberculosis produces no exotoxins and does not contain
endotoxin in its cell wall. In fact, no mycobacteria produce toxins.
 M. tuberculosis can survive within inactivated macrophages.
 Because Activated macrophages can kill the bacteria.
 Individual’s immunological response determines the outcome of
exposure.
 Healthy individual who are exposed to low dose
→ activated macrophages stop infection.
 Individuals unable to mount a rapid response
→ bacteria multiply in lung macrophages.
→ phagocytes attracted to site of infection
→ infection may be walled-off/ forms tubercle.
Tubercles may calcify and become visible in chest X-rays
(Ghon complex)

19. GHON COMPLEX

21. Macrophage engulfing M. tuberculosis pathogen

24.  Bacteria in tubercles may survive for decades (latency).
 Suppression of immune system may allow bacteria to break out
of lesions and multiply (reactivation).
 Old age, cancer, immunosuppressive drugs and HIV infection
can lead to reactivation.

25. Multi-organ Involvement:

26. SIGNS AND SYPTOMS:
 During Active TB symptoms of TB are present, which can include:
 coughing
 weight loss
 loss of appetite
 night sweats
 fever
 chest pain
 Blood stained
sputum
 Active TB is infectious and can be spread by coughing, sneezing,
laughing, singing, or just talking.

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

28.  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
Renal tuberculosis Urine
Tuberculous meningitis CSF
LABORATORY DIAGNOSIS
Specimen Collection:

29. DIAGNOSTIC METHODS

30. i) Direct Microscopy:
 Ziehl-Neelsen staining(hot staining method):
With Ziehl-Nielsen stain, M. tuberculosis look slender, straight or slightly
curved rod with beaded appearance.

31. Kinyoun’s method (cold staining method):
Acid fast bacilli resist decolourisation with acid and alcohol once
they have been stained with carbolfuchsin. The bacilli appear as
pink, long, slender bacilli with beaded appearance.

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

33. ii) Culture:
 Concentrated specimen is generally inoculated on Lowenstein – Jensen’s
medium (solid medium) and incubated at 370
C for 2 – 8 weeks.
 Lowenstein – Jensen’s medium contains coagulated egg, Mineral salt solution,
Asparagine's, Malachite green, Agar
 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.

34. Colonies of Mycobacterium tuberculosis on
Lowenstein-Jensen medium.

35. M. tuberculosis bacterial colonies:

36. Eight Week Growth of Mycobacterium
tuberculosis on Lowenstein-Jensen Agar

37. On Liquid Media:
More sophisticated equipment is required
Faster detection of growth
Higher sensitivity than solid media
Can also be used for drug-susceptibility testing
Two examples of liquid media used:
–BACTEC
–MGIT
Appear as long serpentine cords
in liquid medium .

38. iii) Allergic Test:
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 (Purified Protein Derivative) is injected
intradermally on flexor aspect of fore arm.
Site is examined after 48 – 72 hrs.

39.  Induration of 10 mm or more is considered positive.
 positive test leads to red area at injection site

40. iv) 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.

41. v) 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

42.  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

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

44. PROPHYLAXIS:
1. General measures:
Adequate nutrition, good housing and health education are as
important as specific antibacterial measures.

45. 2. Immunoprophylaxis:
The BCG (Bacille Calmette-Guerin) vaccine (0.1 ml),
administered soon after birth by intradermal Injection which may be
given at any time during the first year of life.
BCG is live attenuated strain derived from M. bovis → stimulates
development of hypersensitivity to M. tuberculosis.
Albert Calmette Camille Guérin

46. 3. Chemoprophylaxis:
Essential Drug
(Abbreviation)
Recommended Daily Dose in
mg/kg body weight (range)
Isoniazid (H)
Adults: 5 mg (4-6) kg/d, 300mg/d
maximum
Children: 10-15 mg/kg/d, 300
mg/d maximum
Rifampicin (R)
Adults: 10 mg (8-12), 600mg/d
maximum
Children: 10-20 mg/kg/d, 600
mg/d maximum
Pyrazinamide (Z) 25 mg (20-30), 2000 mg/d
maximum

47. Essential Drug
(Abbreviation)
Recommended Daily Dose in
mg/kg body weight (range)
Ethambutol (E) Adults: 15 mg (15-25), 1600 mg/d
maximum
Children: 20 mg/kg (range 15-25
mg/kg) daily
Streptomycin (S) 15 mg (12-18)
Maximum for <40 years = 1g
Maximum for ≥ 40 years = 0.75g

48. DOTS – Directly Observed
Therapy Short Course
 DOTS is the name given to the World Health
Organization-recommended tuberculosis
control strategy. DOTS has five main components:
1. Government commitment (including political) at
all levels, and establishment of a centralized and
prioritized system of TB monitoring, recording and training).
2. Case detection by sputum smear microscopy.
3. Standardized treatment regimen directly of six to nine months observed by a
healthcare worker or community health worker for at least the first two months.
4. A drug supply.
5. A standardized recording and reporting system that allows assessment of
treatment results.
MDR TB (Drug Resistant TB)
MDR-TB refers to strains of the bacterium which are proven in a laboratory to be
resistant to the two most active anti-TB drugs, isoniazid and rifampicin. Treatment
of MDRTB is extremely expensive, toxic, arduous, and often unsuccessful.
DOTS has been proven to prevent the emergence of MDR-TB, and also to reverse
the incidence of MDR-TB where it has emerged.

50. THANK YOU