1. بسم الله الرحمن الرحيم

2. Mycobacteria By
Prof. Fouad Elkenawy

3. Mycobacteria Obligate aerobe. Rods. Non spore forming .
Non capsulated .
Acid fast bacteria .

4. Mycobacteria
Cell wall contains high lipid content ( %), which is responsible for their staining &cultural characters .
Not classified as either Gram-positive  or Gram-negative as they do not have the chemical characteristics of either.
Acid fast Stained by Ziehl- Neelsen stain.

5. Classification 1.Members of M. tuberculosis.
Cause tuberculosis in human .
(M tuberculosis, M bovis. )
2. Non tuberculous species ( Atypical mycobacteria ) .:-
Virtually all other species :- Examples :- M.Avian intracellulare ;. M.kansasii ;M. scroflaceum.
3..M.leprea : Cause Leprosy in man .
The causative agent of leprosy.  

6. Mycobacterium tuberculosis
The etiologic agent of tuberculosis (TB) in humans.
* 33 % world wide infetion .
Humans are the only reservoir for the bacterium.

7. General Characteristics of the organism .
Facultative intracellular parasite, usually of macrophages .
High lipids ; 60 % content in the cell wall of M. tuberculosis causes :
Impermeability to Ordinary stains & dyes .
Resistance to many antibiotics .
Resistance to acids & alkalis . Stained by strong basic dyes using heat . Once stained it resist decolorization even usin g strong acids and alcohol so they called acid and alcohol fast .

8. Mycobacteria. Different types of structure
Morphology
Small , straight or slightly curved bacilli , non motile , non sporulated .
Occur singly, in pairs or in masses .
Acid & alcohol fast (resist decolurization with acid & alcohol) .
Not stained by Gram .
Mycobacteria. Different types of structure

9. Cultural characters
Obligate aerobe .
5 to 10% CO2 , enhances growth .
No growth on ordinary media.
Optimum temperature is 37 ºC (strict mesophile).
No growth below 30 ºC or above 39 ºC .
Very slow grower, may require 4 to 6 weeks to get visual colonies

10. Media used to grow M.TB An egg based medium An agar based medium
Lowenstein-Jensen
Middle brook's
Media used to grow M.TB
An egg based medium
Lowenstein-Jensen & Dorest egg media .
An agar based medium
Middle brook's medium .

11. Tuberculosis The source : ishuman and transmited by respiratory droplets . Which inhaled to settle in the alveoli .
Affects the lower respiratory system .
Characterized by:
-Chronic productive cough.
-Low-grade fever.
-Night sweats .
-Weight loss.

12. Pathogenesis
Tuberculous mycobacteria enter the alveoli by airborne transmission .
They resist destruction by alveolar macrophages & multiply, forming the primary lesion or tubercle( gohans focus ).
They then spread to regional lymph nodes ( gohons triad ), enter the circulation, & reseed the lungs.
Tissue destruction results from cell-mediated hypersensitivity.

13. Pathogenesis

14. Host Defenses
Acquired resistance is mediated by T lymphocytes, which lyse infected macrophages directly or activate them via soluble mediators (e.g gamma interferon) to destroy intracellular bacilli.
Antibodies play no protective role .

15. Diagnosis of pulmonary tuberculosis

16. Clinical specimens Sputum, bronchial or gastric washings.
At least 3-5 morning samples of sputum are required for diagnosis .

17. carbol fuchsin
fluorochrome
2.Direct smear
Prepared from sputum either direct or after liquefaction by N-acetyl –L cysteine or by other methods .
Stained by carbol fuchsin (Ziehl-Neelsen) or fluorochrome stain .
By Ziehl-Neelsen(Z-N) stain ,acid-fast bacilli appear pink against blue background.
By fluorochrome stain , the bacilli appear as bright yellow fluorescent against dark background .

18. Acid fast bacilli , Z-N stain

19. Acid fast bacilli, Z-N stain

20. Acid fast bacilli, Z-N stain

21. Advantages of direct smear
Rapid
cheap
Simple

22. Disadvantages of direct smear
Non sensitive ,the sensitivity range is to 75%.
At least ,000 bacilli per ml of sputum are needed to detect positive smear .
Non specific ; Other acid-fast bacilli are similar to M.TB. in stained smear & should be ruled out by other methods .
A positive stain & negative culture may be caused by non viable organisms, which can occur in persons receiving anti tuberculous medication.

23. 3. Culture
Sputum sample is treated  with NaOH , to kill other contaminating bacteria (decontamination ) but does not kill the M.TB. because M.TB. is resistant to alkali .
Sputum is inoculated in selective medium containing antimicrobial agents as (Lowenstein-Jensen ).
Cultures are incubated at 35°C to 37°C in an atmosphere of 5 to 10% CO2.

24. Advantages All cultures should be examined weekly for 4- 8 weeks
Colonies on L-J medium are, raised , rough , confluent, grayish & dry (eugenic growth) .
Advantages
Culture is highly sensitive & specific .
Allows visualization of colony morphology & pigmentation, which is useful for distinguishing colonies of M.TB from those of some non tuberculos mycobacteria .

25. Colonies of M.TB. on L-J medium

26. Colonies on L-J medium

27. Rapid culture method (BACTEC System)
The media used are broth media containing radio-labeled palmitic acid as the sole carbon source.
As M.TB. multiplies, it utilizes the palmitic acid & release radio-labeled CO2.
Using the BACTEC system, M.TB. growth can be detected in 9-16 days .

28. 4.Intradermal skin test (Tuberculin test)
Priciple
Individual previously infected with tubercle bacilli will develop hypersensitivity to proteins of M.TB. which develops 6-8 weeks after infection.
Intradermal injection of PPD into a previously infected, hypersensitive person results in the delayed (48-72 hr) appearance of an indurated reaction.

29. Tuberculin test
Performed by using old tuberulin or PPD, injected either intradermal or by multipuncture
Mantoux test is the standard tuberculin test .
It requires the intradermal injection of (0.1 ml) containing (5 tuberculin units) of PPD .
The transverse diameter of induration is measured 48 to 72 hours later .

30. Method of Tuberculin test

31. Measurment of Tuberculin test

32. Tuberculin test (Interpretation)
Positive Diameter of the induration is 10 mm or greater There is erythema , swelling & induration Positive reactions means previous exposure to M.TB. Negative reaction Induration less than 5 mm .

33. Tuberculin Test
It is impossible to distinguish between active disease & past infection by a positive tuberculin test.
Recent conversion of the reaction from negative to positive needs clinical attention.

34. False positive tuberculin tests
Positive reaction in absence of previous exposure to M.TB .
May be due to prior exposure or infection with other mycobacteria or vaccination with BCG.

35. False negatives tuberculin tests
Negative reaction in spite of previous exposure to M.TB .
Occurs in persons with impaired CMI response & other conditions such as late stages of tuberculosis , malnutrition & steroid therapy

36. Recent methods for diagnosis
DNA probes and gas-liquid chromatography Rapid, specific & sensitive methods for identification of mycobacteria after sufficient growth is present on medium.
PCR :
Useful for direct detection of mycobacteria in clinical specimen within 24 hours or less .
It is rapid, specific & sensitive .

37. Diagnosis of renal tuberculosis
Sample: early morning urine of at least 3 successive days .
Centrifugation of urine &preparation of smear from the deposit .
Staining of the smear by Z-N stain , decolourized by both acid &alcohol to differentiate M.TB from M.smegmatis which is found normally in the genitalia & is acid fast but non alcohol fast .
Culture of deposit after decontamination on L-J medium .

38. Diagnosis of tuberculous meningitis
C.S.F is collected by lumbar puncture .
C.S.F is less turbid than that in meningococcal meningitis & contains excess of lymphocytes
Direct film is prepared from the deposit after centrifugation (Diagnostic) .
The deposit is cultured directly on L-J medium or Dorest egg medium without decontamination .

39. Mycobacterium bovis
Similar to M.TB in morphology & culture characters.
Differs in being shorter & thick .
On L-J medium , gives poor growth as glycerol does not favour its growth (dysgonic) .
Pyruvic acid favours its growth.

40. Mycobacterium bovis Causes TB in cows & in humans.
Both cows & humans can serve as reservoirs.
Humans can be infected by consumption of non pasteurized milk.
This may lead to the development of extra pulmonary TB .

41. Treatment
Tuberculosis is usually treated with  four different antimicrobial agents .
The course of drug therapy  usually lasts from 6-9 months.
The most commonly used drugs are rifampin , isoniazid ,pyrazinamide , & ethambutol , or streptomycin .

42. Prevention of tuberculosis

43. BCG vaccine
Living attenuated vaccine prepared from bovine strain ( bacillus of Calmette & Guerin) .
Prepared by repeated subculture of bovine strain for 250 times on glycerol-potato-bile medium .
Given by intradermal injection .

44. BCG vaccine
Given to all infants in the first year of life & to tuberculin negative adults .
BCG vaccinated person will be converted from negative tuberculin reactor to positive reactors (tuberculin conversion) .
Should not be given to tuberculin positive persons .

45. Non Tuberculous MycobacteriaBy
Prof. Fouad Elkenawy

46. Non tuberculous Mycobacteria
Previously referred to as "atypical" mycobacteria
Widely distributed in natural sources .
Non virulent to guinea pigs .
Not transmitted from human to human, but is acquired from natural sources .
Are important opportunistic pathogens in immuno compromised patients .

47. Non tuberculous Mycobacteria Causes
pulmonary disease by M kanssasii and M avium-intracellulare,
Disseminated infection :In immunocompromised patients, caused by the M avium-intracellulare complex
Cervical lymphadenitis by M scrofulaceum
Granulomatus skin lesions & soft tissue infections by M marinum (swimming pool granuloma) or M ulcerans.

48. Non tuberculous Mycobacteria
Resistant to the usual anti -tuberculosis drugs.
No vaccine is available.
Diagnosis requires culture & identification.

49. Classification
Non tuberculous mycobacteria are classified by pigmentation in the light or dark & by growth rate into 4 groups (The Runyon groups).
Species in groups I to III are slow growers further characterized by pigment production

50. The Runyon groups
Group I : Photochromogens
pigmented only when exposed to light.
Group II: Scotochromogens
Form pigment in the dark.
Group III: Non chromogens are non pigmented .
Group IV : are rapid growers.

51. M.tuberculosis Character
Differences between M.tuberculosis & Nontuberculous Mycobactemria
Character
M.tuberculosis
Non tuberculous mycobacteria
Acid fast
Growth on L-J medium
grow
Temperature
Strict mesophile
Grow at 22,37 & 45 ºC .
Growth
Slow
Slow or rapid
Pigment
Non chromagen
Non chromagen,photo,
or scoto chromagen
P-nitrobenzoic acid
Inhibit growth
No effect .
Pathogenicity to guinea pig
Pathogenic
Non pathogenic
Anti tuberculous drugs
Sensitive
Resistant
Transimmion
Human to human
Not
Human infection
Tuberculosis
lymphadenitis ,skin
&pulmonary

52. Mycobacterium leprae By
Prof. Fouad Elkenawy

53. Mycobacterium leprae
Causes leprosy.
Humans are the only natural host for M.leprea .
Structure M. leprae is similar to other mycobacteria

54. Mycobacterium leprae
Morphology: acid fast bacilli ,found in large masses or single, straight or slightly curved . Strict aerobic ;prefer cool temoerature 30 c . So it infect superficial cool areas . Skin and nerves .
Stained by modified Ziehl- neelsen stain which is modified by using 5% H2So4 for decolourization, It is less acid fast than M.TB .
Cannot be cultivated in vitro & it multiplies very slowly in vivo .

55. Clinical Manifestations
Two clinical forms
1.lepromatous
Granulomatus nodules in skin , peripheral nerves, mucous membranes & organs. ( Leonine face )
Loss of specific cell-mediated immunity.
2.Tuberculoid :- ( Claw hand ) .
Skin and nerve lesions .
Intact cell-mediated immunity .

56. Diagnosis Specimens Nerve , skin lesions biopsies & nasal scrapings .
Film
Stained by modified Z-N stain to show acid fast bacilli in masses or single .
Culture
M .leprae cannot be cultured .

57. The lepramin skin test The PCR technique
A heat-killed suspension of M leprae is injected into skin of the patient
Has little diagnostic value but will provide information of prognostic importance about the immune status of the individual.
The PCR technique
By which very small amounts of M leprae DNA can be detected directly in clinical specimens . May be useful in diagnosis .

58. Thank You