This document provides information on the genera Mycobacterium and Actinomycetes. It describes the characteristics and laboratory diagnosis of key mycobacterial species like M. tuberculosis and M. leprae which cause tuberculosis and leprosy, respectively. It outlines the stages of primary tuberculosis and differences between primary and post-primary disease. It also discusses Actinomyces israelii and Nocardia asteroides, including their morphology, diseases caused, and treatment.

1. ‫بسم ا الرحمن الرحيم‬
GENUS: MYCOBACTERIUM
Prof. Khalifa Sifaw Ghenghesh

2.  Obligate aerobe, Gram-positive rods
 Acid fast
 Complex cell wall lipids
– include mycolic acids
– protects vs. phagolysosomal components
 Peptidoglycan, glycolipids
– acid-fastness
 Two major groups:
– Slow growers:
– Rapid growers:

3. Mycrobacterium tuberculosis
 Non-motile, Non-sporing, non-capsulate
rods
 Grows on several enriched culture
media:
– Lowenstein-Jensen medium:
 Whole
egg, Glycerol, Asparagine, Mineral salts,
Malachite green
Mycobacterium bovis:

4. Lowenstein-Jensen Plate Culture Inoculated
with 15 Strains of Mycobacterium Species

5. CULTURE CHARACTERISTICS
 On primary isolation:
– visible growth after up to 8 weeks
 Colonies:
– Buff colour, dry bread crumb-like appearance
– Growth is eugonic (M. bovis = dysgonic)
 Growth temperature:
– 35-37oC
 Obligate aerobe
-------------------------------------------------------------------------- Heat-sensitive
 Susceptible to alcohol, glutaraldehyde and
formaldehyde.

6. Some differential characteristics of
tuberculle bacilli causing human disease
____________________________________________________
Species
Atmospheric
preference Nitratase TCH Pyrazinamide
--------------------------------------------------------------------------------------M. tuberculosis Aerobic
+
S
S
M. bovis
Microaerophilic
--
R
R
_______________________________________
TCH = thiophen-2-carboxylic acid hydrazide
S = sensitive, R = resistant

7. THE DISEASE
 Not highly contagious:
– transmission with prolonged contact
between susceptible and active case
– usually transmitted by airborne droplets,
must penetrate deep into respiratory tree
– infection can be via other routes:
ingestion
=> infection through cervical
or mesenteric LN

8.  Virulence
– Ability to Survive within Macrophages
 Primary Tuberculosis
 Post-Primary Tuberculosis

9. Stages of Primary Tuberculosis in Childhood
--------------------------------------------------------------------------------------Stage
Time (from onset)
Characteristics
--------------------------------------------------------------------------------------1.
3-8 weeks
Primary complex (PC)
develops and tuberculin
conversion occurs
2.
2-6 months
Progressive healing of PC,
possibility of pleural effusion
3
6-12 months
Possibility of miliary or
meningeal tuberculosis
4
1-3 years
Possibility of bone or joint
tuberculosis
5
3-5 years
Possibilty of genito-urinary or
chronic skin tuberculosis

10. Main differences between primary and post-primary
tuberculosis in the non-immunocompromised patients
--------------------------------------------------------------------------------Characteristics
Primary
Post-primary
--------------------------------------------------------------------------------Local lesion
Small
Large
Lymphatic involvement Yes
Minimal
Cavity formation
Rare
Frequent
Tuberculin reactivity
Negative (initially) Positive
Infectivity
Uncommon
Usual
(pulmonary cases)
Site
Any part of lung Apical region
Local spread
Uncommon
Frequent
---------------------------------------------------------------------------------------

11. TUBERCULIN TEST
 Tuberculin: a heat-concentrated filtrate of a
broth in which tubercle bacilli had been grown.
 Injection of tuberculin into the skin >>
– Large, indurated reactions >>Post-Primary
Tuberculosis.
– No induration >> Protective immunity
 Purified Protein Derivatives (PPD):
– Mantoux Method (Intracutaneous)
– Heaf Method (Spring-loaded gun)
– Tine Tests (Disposable single tests)

12. Mycobacteria-Positive PPD

13. LABORATOY DIAGNOSIS
1. Specimen:
– Pulmonary tuberculosis: > Sputum, Bronchial
washings, Laryngeal swabs, and Early-
morning gastric aspirates.
– Homogenized tissue biopsies.
– Examine after centrifugation: Deposits of
CSF, Pleural fluid, Urine and other fluids

14. 2. Microscopy:
– Ziehl-Neelsen Stain
– Fluorescent dyes
3. Culture:
– Decontamination:
– Lowenstein Jensen medium
4. Nucleic Acid Methods:

15. Mycobacterium tuberculosis sputum
smear (Ziehl-Neelsen stain)

16. Mycobacterium tuberculosis in a
sputum smear (Ziehl-Neelsen stain)

17. Mycobacteria - Auramine Stained and Viewed with
Fluorescence Microscopy.
Acid Fast Bacilli Appear as Glowing Yellow Rods.

18. Histopathology of Tuberculosis, Endometrium.
Ziehl-Neelsen Stain.

19. An anteroposterior X-ray of a patient
diagnosed with advanced bilateral pulmonary
tuberculosis.

20. TREATMENT
Chemotherapy Recommended by International Union against
Tuberculosis and Lung Disease (examples)
--------------------------------------------------------------------------------------------------------Intial phase
Continuation
Regimen
(2 months)
phase (4 months)
Drug
Drug
--------------------------------------------------------------------------------------------------------HRZ
HR
Standard
HRZ
H3R3
Intermittent/ when
HRZ
H2R2
supervision is indicated
HRZE
HR
When there is a high
HRZS
HR
incidence of initial drug
resistance
---------------------------------------------------------------------------------------------------------

21. EPIDEMIOLOGY
 Transmission:
– Open Pulmonary Tuberculosis
– Crowdness in homes and workplaces
 Tuberculosis and AIDS
 Tuberculosis in Developing Countries:
– Africa

22. CONTROL
 Early Detection and Treatment of Open
Cases
 Reducing Overcrowding
 Vaccination:
– Bacille Calmette-Guerin (BCG)
– Not effective as control measure

23. Mycobacterium leprae
 Leprosy (Hansen's disease)
– A chronic intracellular infectious disease
unique to man (with few exceptions).
Usually not fatal.
 Never been cultivated in vitro
 Armadillos:
– 1010 bacilli/gram of diseased tissue

25. M. leprae can be grown in mouse foot pads
or the nine-banded armadillo (picture).

26. PATHOGENESIS
 Schwan cell >> Nerve damage >>
Anaesthesia and Muscle paralysis >>
Gradual destruction of extremities
> Nasal bones and eyes
Immune reactions >>
– Severe and permanent nerve damage

27. THE DISEASE
 Manifestations of the disease depend on the
resistance of the host.
1. Tuberculoid: host is highly resistant,
clinical abnormalities limited to a few
peripheral nerves and adjacent skin areas,
tuberculoid granuloma
2. Lepromatous: host lacks resistance, all
tissues affected, foam cell granuloma
3. Borderline:

28. The feet become subject to bone damage and
deformity through unnoticed wounds and infection.
Serious infections can lead to amputations.

29. Victims of leprosy often suffer amputations of
fingertips and toes due to unfelt trauma.
X-rays of different stages.

30. Leprosy affects facial nerves > loss of blinking
reflex of the eye > dryness, ulceration, and
blindness.

31. Lepromatous lesions on human back

32. LABORATORY DIAGNOSIS
 Histological Examination of Skin Biopsies
 Detection of Acid-Fast Bacilli:
– In Nasal Discharges
– Scrapings from Nasal Mucosa
– Slit-Skin Smears
Superficial incisions in skin >>
Bacillary Index (BI):

33. Bacillary Index (BI)
 1+
 2+
 3+
 4+
 5+
 6+
1-10 bacilli/100 fields
1-10 bacilli/10 fields
1-10 bacilli/
field
10-100 bacilli/ field
100-1000 bacilli/ field
>1000 bacilli/ field
 Morphological Index (MI):

34. TREATMENT
WHO Recommendations for Multidrug Therapy
----------------------------------------------------------------------------------------------Type of
Drug
Dose Frequency
Total
Leprosy
(mg)
Duration
-----------------------------------------------------------------------------------------------
Paucibacillary
Rifampicin
Dapsone
600
100
Monthly, Superv.
Daily, unsuperv.
6 months
Multibacillary
Rifampicin
Dapsone
600
100
300
Monthly, Superv.
Daily, Unsuperv.
Mothly, Superv.
>2 years
+50
Daily, Unsuperv.
Clofazimine
{
-----------------------------------------------------------------------------------------------

35. EPIDEMIOLOGY
 Transmission:
– Nasal secretions of patients with lepromatous leprosy.
 Skin Test: Limited diagnostic value
– Lepromins > boiled-bacilli rich lepromatous lesions
– Leprosins > ultrasonicates of tissue-free bacilli from
lesions.
 Two Types of Reaction:
– Fernandez Reaction: sensitized individuals > 48h
(leprosin)
– Mitsuda Reaction: granulomatous swelling > ~3 weeks
(lepromin)

36. ‫بسم ا الرحمن الرحيم‬
‫‪ACTINOMYCETES‬‬

37.  Gram +ve, Filaments that Break Up
Into Bacillary and Coccoid Forms.
 Non-Motile, Non-Sporing, Non-
Capsulated.
 Free Living >> Soil

38. 1. ACTINOMYCES SPECIES
 A. israelii
– Actinomycosis >>
Chronic Granulomatous Infection.
– Formation of Sulphur granules:
 3 Forms:
i. Cervicofacial
ii. Thoracic
iii. Abdominal
 Predisposing Factors:
Trauma, Poor Oral Hygiene.

39. A. israelii
Gram stain showing diphtheroidal rods and
short branching filaments.

40. Actinomycosis-organisms aspirated from the lung. Long,
tortuous and branching organisms can easily be visualized
using silver stain. Sulfur granules not seen in aspirations.

41. LABORATORY DIAGNOSIS
i. Direct Examination:
Sputum, Pus, etc.. >> Examined
for Granules
ii. Culture: Brain Heart Infusion Agar
TREATMENT
– Penicillin >> Several Weeks

42. Sulfur granule from human actinomycosis tissue
section (hematoxylin and eosin stain).

43. 2. NOCARDIA SPECIES
 N. asteroides
– Nocardiosis.
 Aerobic.
 Disease Begins as Pulmonary Infection >
 > 50% of Patients are Immunocompromised.
 Fatality Rate >>

44. LABORATORY DIAGNOSIS
i. Direct Examination:
 Sputum, Skin Lesions, Tissue Biopsies or Surgical Material
>> Microscopically.
 Observe: G+ve, Multiple Branched and Beaded Filaments.

> Partially Acid-Fast.
 ii. Culture:
 iii. Identification: Biochemically.
TREATMENT
 Sulphonamides, NA, TMP-SMX.

45. Nocardia asteroides
Silver stain showing the twisted masses of
long filamentous organisms

46. Nocardia asteroides
Acid fast stain shows the pale red staining
organisms in an area of necrosis

47. Differences between the genera
Actinomyces and Nocardia
-----------------------------------------------------------------Actinomyces species
Nocardia species
-----------------------------------------------------------------Facultative anaerobes
Grow at 35-37oC
Oral commensals
Non-acid-fast mycelia
Endogenous cause of
Disease
Strict aerobes
Wide temp range of growth
Environmental saprophytes
Usually weakly acid-fast
Exogenous cause of disease
------------------------------------------------------------------