1. MYCOBACTERIA
TUBERCULOSIS (MTB) Mary Mwinga

2. Tuberculosis
• Tuberculosis- Is an Ancient Disease
-Spinal Tuberculosis in Egyptian Mummies
- History dates to 1550 – 1080 BC.
• Robert Koch
- Discoverer of Mycobacterium Tuberculosis (Koch’s disease)
• M. tuberculosis
• major human disease
– Affects healthy people
• problems
– association with AIDS
– multiple drug-resistance
– Chronic disease
– Prolonged treatment

3. What are Mycobacteria?
• Obligate aerobes growing most successfully in tissues with a high
oxygen content, such as the lungs.
• Facultative intracellular pathogens : infect mononuclear
phagocytes (e.g. macrophages).
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4. Classification of Mycobacteria
1. Tubercle bacilli
a) Human – MTB
b) Bovine – M. bovis
c) Murine – M. microti
d) Avian – M. avium
e) Cold blooded – M. marinum
2. Lepra bacilli
a) Human – M. leprae
b) Rat – M. leprae murium
3. Mycobacteria causing skin ulcers
a) M. ulcerans
b) M. belnei
4. Atypical Mycobacteria (Runyon
Groups)
a) Photochromogens
b) Scotochromogens
c) Nonphotochromogens
d) Rapid growers
5. Johne’s bacillus
M. paratuberculosis
6. Saprophytic mycobacteria
a) M. butyricum
b) M. phlei
c) M. stercoralis
d) M. smegmatis
e) Others

6. 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, Non-capsulated.
• Growth generally slow
• Genus includes
• Obligate parasites
• Opportunist pathogens
• Saprophytes

7. Mycobacterium tuberculosis complex
• Includes Human and Bovine mycobacterium
• M .Africanism Tropical Africa
• M. microti do not cause human infections but in small mammals
M. bovis
• Primarily infection among the cattle
• M.bovis infects Tonsils, Cervical nodes, can produce Scrofula.
• Enter through Intestines – infects the Ileocecal region.
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8. What are atypical Mycobacterium
• Infects birds, cold blooded animals worm blooded animals
• Present in environment
• Opportunistic pathogens
• Others – Saprophytic bacteria
-M butryicum present in butter
-M. phlei
-M smegmatis – present in Smegma
8

9. Atypical Mycobacterium
• 1 Photochromogens
• 2 Scotochromogens
• 3 Non Photochromogens
• 4 Rapid growers
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10. Mycobacterium tuberculosis
Epidemiology
• 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.

11. Mycobacterium tuberculosis (MTB)
• Morphology –Straight or slightly curved rods
• 3 x 0.3microns
• May be single, in pairs or in small clumps
• On conditions in growth appears as filamentous,
club shaped, or in Branched forms.

12. Mycobacterium tuberculosis (MTB)
• 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

13. MTB : Cultural characters
• Slow-growing: generation time of 12 to 18 hours (c.f. 20-30 minutes
for Escherichia coli).
• Colonies appear after 2 weeks or at 6-8 weeks
• Hydrophobic with a high lipid content in the cell wall.
• Because the cells are hydrophobic and tend to clump together, they are
impermeable to the usual stains, e.g. Gram's stain.
• MTB - Obligate aerobes.
• MTB grows more luxuriantly than M. bovis.
• Addition of 0.5% Glycerol supports growth of human strains.
• No effect or inhibitory effect on bovine strains.

14. 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

15. Resistance
• Not specifically resistant to heat: destroyed by 60° C x 20 min.
• 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

16. Biochemical reactions
• Niacin test – Human MTB produces niacin when grown in egg
medium.
• Aryl Sulphatase test – Enzyme Aryl sulphatase formed by only
atypical mycobacteria.
• 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 weakly positive.
- MTB is strongly peroxidase positive while atypical mycobacteria
are negative.
• Nitrate reduction test – Positive in MTB and negative in M.
bovis

17. Tuberculosis
• Tuberculosis (TB) is a contagious disease. Like the common cold
spreads through the air.
Only people who are sick with TB in their lungs are infectious.
M.O.T: cough, sneeze, talk or spit propels TB germs (bacilli) into the air.
INFECTION: inhaling a small number of bacilli.
Clinical picture :
* Low grade fever
* Weight loss
* Night sweats
* Fatigue
* Cough & hemoptysis

18. Tuberculosis highly Communicable Disease.
• 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.
• People with HIV and TB infection are much more likely to develop
TB.
In India 1 death / Minute
• Half a million people die from disease every year in India (one death
every minute)
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19. Pathology and Pathogenesis of Tuberculosis
• Source of Infection – Open case of Pulmonary Tuberculosis.
• Has potential to infect 20 – 25 healthy persons before cured or dies
• Through Coughing , Sneezing, or Talking.
• Each act can spill 3000 infective nuclei in the air,
• Infective particles are engulfed by Alveolar Macrophages.
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20. Generation of Droplet Nuclei
• One cough produces 500 droplets
• The average TB patient generates 75,000
droplets per day before therapy
• This falls to 25 infectious droplets per
day within two weeks of effective
therapy
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21. Predisposing Factors
• Genetic basis,
• Age
• Stress,
• Nutrition,
• Co existing infections Eg HIV
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22. 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)

23. Pathogenesis of tuberculosis
1. Pulmonary tuberculosis.
2. Extra-pulmonary tuberculosis.

24. Pathogenesis of tuberculosis
1. Pulmonary tuberculosis
•infects lungs
• distributed within macrophages
• facultative intracellular pathogen
– inhibits phagosome-lysosome fusion
– resists lysosomal enzymes
• other MTB factors:
•Mycobactin - siderophore
• Cord factor - damages mitochondria

25. Pathogenesis
* Inhalation of tubercle bacilli
* They multiply in the alveolar macrophages
* An early tubercle (granuloma) is formed

26. Mechanisms of Infection
• Within 10 days of entry of Bacilli - clones of Antigen specific T
Lymphocytes are produced
• Can actively produce Cytokines, Interferon γ activate Macrophages
to form cluster or Granuloma.
Tubercle with Caseous Necrosis 26
Giant cells
Tubercle bacilli
Partially activated
macrophage
Lymphocyte
Fully activated
macrophage

27. Basis of Tubercle formation (Granuloma).
• Tubercle is an Avascular granuloma Containing central zone of giant
cells with or without caseation and peripheral zone of Lymphocytes
and Fibroblasts.
• Produce lesions may be
Exudative or Productive
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28. Diagram of a
Granuloma
NOTE: ultimately a fibrin layer
develops around granuloma
(fibrosis), further “walling off” the
lesion.
Typical progression in pulmonary
TB involves caseation,
calcification and cavity formation.
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29. Tubercle discharging
Bronchial tree
TNF- aTNF- a
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30. Immunity in Tuberculosis.
• CD4 T Lymphocytes with Th 1 or Th 2 secrete - Cytokines,
Interleukin 1,and 2 , Interferon's γ and Tumor necrosis factor.
• Th 1: secrete Cytokines  Activate Macrophages
Results in protective Immunity, and contain Infection.
• Th 2 manifests with Delayed Hypersensitivity
DTH causes Tissue destruction.
and disease will progress.
.
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31. Immunity in Tuberculosis
• Activated Macrophages - Epithelioid cells Forms cluster a
granuloma
• Activated macrophages turn into Giant cells.
• Granuloma contains: necrotic tissue, dead macrophages, cheese like
caseation.
• Apoptosis of bacteria laden cells contribute to protective immunity.
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32. Lesions in Tuberculosis
• Exudative and Productive.
1. Exudative – Acute inflammatory reaction with edema fluid –
contains Polymorphs- Lymphocytes – later Mononuclear cells.
-Bacilli are virulent - Host responds with DTH Injurious.
2. Productive Type protective Immunity
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33. 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 polymorphonuclear
leucocytes
- Help survival of tubercle bacilli inside
macrophages

34. 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 is immune & hypersensitive

35. Classical tubercular lesion – Granuloma with typical
Langhan’s giant cells, epithelioid cells, lymphocytes and
fibrosis.

36. 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.

37. 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.)

38. Koch’s Phenomenon
• Tuberculosis infected Guinea pig if injected with Living Tubercle
bacilli:
-The site around the injection becomes necrotic.
• Koch found the same reaction when injected with old Tuberculin (
heated and concentration of the tubercle bacilli )
• It has produced the same reaction
• This is called as Koch’s Phenomenon.
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39. Post Primary Tuberculosis
• Due to Reactivation of Latent infection.
• Also due to Exogenous reinfection
• Differs from Primary Infection.
• Leads to –
Cavitation's of Lungs, Enlargement of Lymph nodes,
Expectoration of Bacteria laden sputum
Dissemination into Lungs and other extra pulmonary areas.
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40. Complication of Tuberculosis.
1. Meningitis.
2. Pleurisy,
3. Involvement of Kidney,
4. Spine ( Potts’ spine )
5. Bone Joints,
6. Miliary tuberculosis
40

41. Symptoms and Sings of Tuberculosis
41

42. Clinical Illness with Tuberculosis
• Pulmonary Disease – Major
manifestation with involvement of
Lungs
-Hemoptysis, Chest pain Fever sweets
-Anorexia
-Cavity formation in Lungs
42

43. 2. Extra pulmonary Tuberculosis
• Bacteria on circulation leads to bacteremia leads to involvement of
-GUT, Genito urinary system, Meningitis
-Gastro Intestinal system, skin, Lymph nodes Bone marrow.
-Spinal infection Potts spine, Arthritis
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44. Multidrug-resistant tuberculosis (MDR-TB)
• DEF: MDR-TB is resistance to isoniazid and rifampicin, with or
without resistance to other first-line drugs (FLD).
• XDR-TB is resistance to at least isoniazid and rifampicin, and to any
fluoroquinolone, and to any of the three second-line injectables
(amikacin, capreomycin, and kanamycin).
• Drug resistance is due to improper use of antibiotics in
chemotherapy of drug-susceptible TB patients.
• As a result of a number of actions including, administration of
improper treatment regimens and failure to ensure that patients
complete the whole course of treatment.
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45. Laboratory diagnosis M. tuberculosis
• Acid fast bacteria demonstration in sputum smear.
• Culture on L J media
• Biochemical identification
• Antibiotic sensitivity test
• Skin testing
• delayed hypersensitivity
• tuberculin
• protein purified derivative (PPD)
• X-ray
• PCR

46. SPECIMENS
* According to site of infection :
- Sputum - Urine - Body fluids
- Gastric lavage - Blood - Tissue biopsy
* Specimens need appropriate processing
Liquefaction with N-acetyl-L- cysteine
Sputum Decontamination with NaOH
Centrifugation

47. Laboratory Diagnosis
• Pulmonary TB –
• Specimen – ZN
• 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
Advantage: - cheap – rapid
- Easy to perform
- High predictive value > 90%
- Specificity of 98%
Disadvantages:
- sputum ( need to contain 5000-10000 AFB/ ml.)
- Young children, elderly & HIV infected persons may not produce cavities & sputum
containing AFB.

48. Acid fast bacilli
48

49. Detecting AFB by fluorochrome stain using fluorescence microscopy:
• The smear is stained by auramine-O dye.
• TB bacilli are stained yellow against dark background & easily visualized
using florescent microscope.
Advantages:
- More sensitive
- Rapid
Disadvantages:
- Hazards of dye toxicity
- more expensive
- must be confirmed by Z-N stain

50. Acid fast Bacilli seen as in Florescent Microscope
50

51. Quantitation of AFB in Sputum Smears
• No of Bacilli No of Fields Report as
• 0 300 Negative
• 1-2 300 Doubtful
• 1- 9 100 1+
• 1- 9 10 2+
• 1-9 1 3+
• 10 or >10 1 4+
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52. CULTURE:
Nature of Growth Characters
• M tuberculosis is obligate aerobe.
• M.bovis Microaerophilic
• M.tuberculosis growth luxierently
• M.tuberculosis eugonic
• M bovis is dysgonic
• When grown on 0.5% glycerin M tuberculosis growth improves
• Sodium pyruvate improves the growth of both organism.
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53. Culture
Lowenstein Jensen Medium –
- Selective medium.
- Always in screw capped bottle.
- Color: Bluish-Green.
-Contains – Egg protein – Solidifying agent
-Mineral salts – Mg sulphate, Mg citrate
-Asparagine
-Malachite Green – Selective agent
•GROWTH: grows very slowly: several weeks
– non-pigmented colonies
– niacin production: differentiates from other
mycobacteria
Sterilized by - Inspissation

54. On L J Medium
• MTB appear as dry, rough raised irregular colonies.
-Appear wrinkled, creamy white
-Become yellowish.
• M. bovis appear as flat smooth, moist, white and break
up easily.
• Advantages: - Specificity about 99 %
- More sensitive (need lower no. of bacilli 10-100 / ml)
- Can differentiate between TB complex & NTM using
biochemical reactions
- Sensitivity tests for antituberculous drugs ( St, INH,
Rif., E)
• Disadvantages: Slowly growing ( up to 8 weeks )
54

55. Laboratory Diagnosis
• Extra -Pulmonary TB –
• Specimen –
• CSF – in suspected meningitis
• Pleural fluid & other exudates
• 2-3 days urine in renal TB
• Biopsy material.

56. Concentration methods
• Purpose – Homogenization & Concentration in sputum & other
specimens.
• 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 Cetrimonium bromide 20 g + NaOH 40 g in one
litre. Allow to stand for five minutes. Inoculate Acid Buffered LJ Medium with swab.

57. Antigenic Characters
• Group specificity due to Polysaccharides.
• Type specificity due to protein antigens.
• Delayed hypersensitivity to proteins
• Related to each other species
• Some relation between lepra and tubercle bacilli
• Serology – Tests not useful
Antigenic homogeneity between < bovis and M.microti
57

58. Molecular Typing
• DNA finger printing differentiates different strains
of Mycobacterium species
• Treating the organism with Restriction endonuclease
yields Nucleic acid fragments of varying length and
strain specific
• Used in epidemiological studies
58

59. Finger printing Methods
• Done with Chromosomal insertion sequence IS 6110
present in most strains of Tubercle bacilli.
• Now entire genome of M. tuberculosis is sequenced
• Several Molecular methods are available for studies
59

60. Tuberculin Test (Mantoux test)
• Delayed hypersensitivity skin test to assay:
-cell mediated immunity to tubercle bacillus
Material: A purified protein derivative (PPD)
Dose : 0.1 ml of (PPD) is injected
intradermal
Reading:
Positive test-
- Induration equal or greater than 10 mm
- Develop 48-72 hours after injection
Negative test: induration less than 10 mm.
• Positive test interpretation:
indicates exposure to organism
 does not indicate active disease

61. 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

62. Tuberculin Testing - Limitations
• 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
62

63. Recent Methods for Diagnosis
I – BACTEC 460 ( rapid radiometric culture system )
-specimens are cultured in a liquid medium (Middle brook7H9 broth
base) containing C14 – labeled palmitic acid & PANTA antibiotic
mixture.
- Growing mycobacteria utilize the acid, releasing radioactive CO2
which is measured as growth index (GI) in the BACTEC
instrument.
-The daily increase in GI output is directly proportional to the rate &
amount of growth in the medium.
63

64. III Polymerase Chain Reaction (PCR) & Gene probe
- Nucleic acid probes & nucleic acid amplification tests in which
polymerase enzymes are used to amplify ( make many copies of
specific DNA or RNA sequences extracted from mycobacterial cells.
Advantages:
- Rapid procedure - High sensitivity (1-10
( 3 – 4 hours) bacilli / ml sputum)
64

65. Tuberculosis and HIV infection
• HIV association has become a threat to the developed countries too
• HIV association will lead to rapid spread of tuberculosis
Considerations:
• HIV is the strongest risk factor for progression to active disease
• HIV kills CD4+ T Helper cells which normally inhibit M. tuberculosis
• HIV interferes with PPD skin test
• Protease inhibitors interfere with rifampin
65

66. MDR tuberculosis
• MDR TB is a global threat.
• In 1993 WHO declared Tuberculosis a Global emergency
• Animals shed the bacilli in Milk, human’s get infected after drinking
the unsterilized Milk
• Pasteurization has reduced the incidence of Bovine tuberculosis.
66

67. TREATMENT
--Drugs - Rifampicin
- Isoniazid isonicotinylhydrazide (INH)
--Bactericidal
-Pyrazinamide
-Streptomycin
-Ethambutol
-Ethionamide
-Thioacetazone/ amithiozone
--Bacteriostatic
-Paraminosalicylic acid
-Cycloserine

68. Treatment
Drugs used :
1- First line drugs :
- Isoniazid - Rifampicin - Pyrazinamide
- Ethambutol - Streptomycin
2- Second line drugs (more toxic and less effective):
- Kanamycin - capreomycin - Cycloserine
- ethionamide - ciprofloxacin - Ofloxacin
* Noncompliance (failure to complete the course):
- Directly observed therapy (DOT)
-Health care workers observe the medication

69. Treatment
• Chemoprophylaxis – INH for one year
- Domicilliary treatment preferred
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

70. 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%

71. 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, leukemias and other malignancies by
non-specific stimulation of RE system.