1. Acid-Fast Bacilli

2. Mycobacterium Group

3. Definition:
Generally, these microorganisms are characterized by their special reaction to stain.
They are difficult to be stained by ordinary stain due to the high content of lipoid substances in the cell wall.
However, once they stained they resist decolorization even with acid, thus they called acid fast bacilli when stained with Zhiel -Neelsen stain.

4. The acid fastness is due to the high lipid content (about 60%) of their cell wall.
They contain N-glycolylmuramic (mycolic) acid instead of N-acetylmuramic acid.
They are Gram +ve but stained poorly.
Non spore forming, non motile and non capsulated.
This group includes both pathogenic and non pathogenic bacteria.

5. Classification Mycobacterium tuberculosis Mammalian type M. leprae
Human.
Bovine.
Murin type.
M. leprae
Atypical Mycobacterium
Avian type
Reptilian or cold blood type
Saprophytic Mycobacterium

6. Mycobacterium tuberculosis (Human or Bovine type)

7. Culture characteristics:
Morphology:
Acid Fast beaded bacilli arranged in bundles.
Non spore forming.
Non motile.
Non capsulated.
Culture characteristics:
Require Dorset’s egg media.
Can grow on in presence of Malachite green (Löwenstein-Jensen media) as selective agent.

9. Biochemical Reactions: Niacin test:
Strictly aerobic.
Grow very slow, thus require 6-8 weeks before discarding the culture as negative.
Biochemical Reactions:
Niacin test:
Positive in human type.
Negative in bovine type.

10. Epidemiology:
Tuberculosis still remains among the most important communicable diseases in the world today with an estimated 50 million active cases of who 3 million will die annually from its effects.
In Egypt, the prevalence rate is about 1.7 % of which two thirds are active cases. The death rate is about 15/100,000.

11. There are mainly two modes of infection; either by droplet (airborne) infection and this is generally by human type or by consumption of contaminated milk or milk products and this is usually by bovine type.
Human type: causes pulmonary tuberculosis mainly by droplet infection from an open case.
Bovine type: is attracted by the ingestion of contaminated milk or milk products.

12. The bacilli will reach to the cervical glands
The bacilli will reach to the cervical glands. Also, the organism may reach to the mesenteric lymph nodes causing lymphadenitis.
From the lymph glands the bacilli may reach to many organs causing generalized miliary tuberculosis.
Primary tuberculosis; may occur at any epithelial site (at any part of the body); however, it is most commonly restricted to the lung TB.

13. Pathogenicity:
Mycobacteria survive after ingestion by macrophages and behave as facultative intracellular organisms.
The infected cells express histocompatibility complex (MHC)-associated bacterial peptides that trigger T cells responses.
Activated CD4+ and TH1 cells release large amounts of interferon- (IFN-), which activates the infected macrophages.

14. The activated macrophages, in turn, destroy the intracellular Mycobacterium.
The major pathogens are Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium leprae, the cause of leprosy.
Atypical mycobacterium, such as Mycobacterium avium-intracellulare complex and Mycobacterium kansasii, can cause tuberculosis disease but less frequent pathogens.

15. Rapidly growing mycobacteria, such as Mycobacterium chelonei, are saprophytes that occasionally cause human disease in immunocompromised hosts.
In human, the tuberculosis is either:
 Pulmonary type.
 Extra-pulmonary type.

16. Determinants of Pathogenicity:
Cording factor is a glycolipid derivative of mycolic acid that is present on the outer surface of M. tuberculosis. The glycolipid inhibits migration of polymorphonuclear (PMN) leucocytes and elicits granuloma formation. Also, it is immunogenic.
Sulfatides permits the bacteria to survive in the macrophages.
Antibacterial resistance; by mutation.

17. Clinical picture:
The typical symptoms include; fever, fatigue, night sweats, and weight loss.
Many organs can be involved. Miliary tuberculosis (spread via blood) is characterized by multiple disseminated lesions that resemble millet seeds.
Tuberculous meningitis and tuberculous osteomyelitis are important disseminated forms.

19. Laboratory Diagnosis:
Direct Methods:
The specimen.
Direct Examination (Zhiel-Neelsen stain).
 Methods Depend on Isolation:
Culture.
Biochemical reactions.
Animal inoculation.
Methods other than isolation:
Tuberculin (Mantoux) Test.
Chromatographic Analysis.
Molecular Methods.

20. Sputum: in case of pulmonary tuberculosis. Pus: in case of skin type.
Direct Methods:
The specimen:
Sputum: in case of pulmonary tuberculosis.
Pus: in case of skin type.
Urine: in case of urinary tuberculosis.
CSF or blood: in case of meningitis.
Blood: in case of miliary tuberculosis.
Stool: in case of intestinal tuberculosis.

21. Special Remarks:
For sputum: It's better to take the morning sample and for 3 successive days. The sample requires decontamination process and may be concentration.
Pus: could be treated as sputum.
Urine: is treated as above and in direct examination acid and alcohol must be used to avoid the false +ve result due to presence of the saprophytic mycobacterium in the smegma.

22. CSF: it's a normal sterile liquid, thus direct examination and culture are done directly from the deposit after centrifugation without the need of decontamination process.
Stool: treated as urine.

23. Direct Examination:
A film is prepared and suspected specimen and is stained with Ziehl - Neelsen stain.
The appearance of acid-fast bacilli having the morphology of TB (tubercle bacilli) is almost diagnostic of tuberculosis infection.
Alternatively, the bacilli could be detected with auramine stain and visualized by fluorescence microscope.

24.  Methods Depend on Isolation:
Culture:
May be positive even when the direct test is negative and this could be attributed to the presence of very few numbers of bacilli in the sample.
The sample (from non sterile sites) is undergone a process of decontamination and concentration.

25. Culture is done on Löwenstein-Jensen or Middlebrook medium and incubates aerobically at 37 C and examined weekly up to 8 weeks before considering it negative.
Colonies are dry and warty appearance and buffy in color (human type) or soft and flat colonies (bovine type).
Alternatively, culture could be made in liquid BACTEC medium, in which radioactive metabolites are used and growth can be detected by the production of radioactive CO2 in shorter time.

27. Biochemical reactions:
Niacin test is +ve in case of human type and -ve in case of bovine type.
Catalase -ve.
Animal inoculation:
The prepared sample for culture may also be injected into two guinea pigs. One of these animals is killed after 4-6 weeks and examined for the typical lesions of tuberculosis and also by film stain and culture. The other animal is left under observation.

28. Methods other than isolation:
Tuberculin (Mantoux )Test:
The immunological base of this test is the type IV hypersensitivity or delayed type which depends on cell mediated immune reaction.
This test was firstly described by Koch who used the crude extract of TB (tubercle bacilli) known as Koch's Old Tuberculin (K.O.T).
Recently, a purified protein derivative (PPD) is used instead.

29. Tuberculin test is performed by intradermal injection of the PPD (5 IU) in one forearm.
In case of +ve tuberculin, a red and indurated area (10 mm in diameter) appears after 2-3 days. The induration is very important.
Significance of tuberculin test:
Tuberculin + ve; means a case or previously exposed to TB or vaccinated. Since by adulthood as many as 80% of Egyptians are positive, thus the test is of great diagnostic value (together with the clinical symptoms) in childhood.

30. Tuberculin -ve; means neither a case nor having immunity.
Tuberculin test is used before BCG vaccination, which should be given only to tuberculin -ve people. If it is given to tuberculin +ve individual, it may provoke a virulent unwanted reaction.

31. Bacillus Calmette–Guérin is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus, Mycobacterium bovis, that has lost its virulence in humans by being specially subculture in an artificial medium for 13 years, and also prepared from Mycobacterium tuberculosis.

32. Albert Calmette
Camille Guérin

33. Chromatographic analysis
Depend on the analysis of fatty acids by Gas chromatography or HPLC.
Molecular Methods:
These methods are based on the detection of a specific gene(s) of the TB. Of these methods:
 DNA probe method (Nucleic acid hybridization).
Nucleic acid probes are commercially available. The detection is done within 2-3 hours.

34. The cost, technical expertise.
 PCR method.
It is a rapid and sensitive method and does not require the presence of the organism but only a specific DNA fragments to be present.
The limitations of these two methods are:
The cost, technical expertise.
The unavailability of the organism to do antibiotic sensitivity testing.

35. Prevention and Control:
B.C.G. vaccine; it is a living attenuated vaccine derived from M. bovis. Attenuation is obtained by repeated subcultring (about 250 times) on unsuitable medium containing glycerol, potato and bile.
Hygienic measures, etc.
Eradication of infected animals (tuberculin +ve) by slaughtering.
Good nutrition and effective pasteurization of milk.

36. Treatment:
Because of drug resistance, antibiotic sensitivity test should be performed.
Resistance by mutation.

37. Atypical or Anonymous Mycobacterium Mycobacterium other than tuberculosis (MOTT)

38. General Features:
Acid fast bacilli differ from the typical TB in being non pathogenic to guinea pig.
They can be treated with antibiotics and they are resistant to anti-tuberculous drugs.
They are widely distributed in the environment.
They form smooth and pigmented colonies.
They are classified according their culture characteristics into:

39. Atypical or Anonymous Mycobacterium
Slow Growing
Rapid Growing
Photochromogenic
Scotochromogenic
Non Chromogenic

40. Slow growing: A. Photochromogenic:
Produce yellow pigment when the growth is being exposed to light.
Example: M. kansasii, M. marinum.
B. Scotochromogenic:
Produce orange color chiefly in the dark.
Example: M. scrofulaceum.

41. C. Non Chromogenic:
Produce no pigments.
Example: M. avium -intracellular complexes.

42.  Rapid growing:
Grow within few days and they can grow in ordinary media.
They can produce several types if infection even mimics TB in symptoms.
Examples:
M. fortuitum -chelonei complex.
M. smegmatis is a rapidly growing mycobacterium that is not associated with human disease. It is part of the normal flora of smegma, the material that collects under the foreskin of the penis.

43. Typical mycobacterium Atypical mycobacterium
Culture
Characteristics
- Produce dry, warty and buff colonies (human type) or soft white colonies (bovine type).
Require enriched media.
Give smooth and pigmented colonies.
Can grow on ordinary media.
Catalase test
-ve
Strongly +ve
Niacin test
+ve (human type)
-ve (bovine type)
+ve
Pathogenicity to guinea pigs
Pathogenic
Non pathogenic
Susceptibility to anti-tuberculous
normally susceptible
Not

44. Mycobacterium leprae (The causative agent of leprosy)

45. Morphology:
They are acid and alcohol fast bacilli when stained with modified Ziehl- Neelsen stain (5% H2 SO4).
They may appear beaded but coarser than TB.
They arrange in masses or groups and mostly intracellular.
Non spore forming.
Non motile.

47. Culture: They cannot grow on any artificial media or cell culture.
It can be grown in the mouse footpad or in the armadillo.
Humans are the natural host.
The optimum temperature for growth is 30 °C. It is lower than body temperature; it thus grows preferentially in the skin and superficial nerves.

49. Epidemiology:
Transmission: Infection is acquired by prolonged contact with patients with lepromatous leprosy, who discharge M. leprae in large numbers in nasal secretions and from skin lesions.
The incubation period is extremely long, lasting from several months to 20 years.
The disease occurs worldwide, with most cases in tropical regions of Asia and Africa.

50. Pathogenicity: Leprosy in man occurs in three clinical forms:
The nodular or lepromatous type:
- In which the organisms produce nodules and form granulation in skin, mucous membrane and internal organs.
Tuberculoid type.
In this case the nerve endings are usually affected with paralysis or loss of sensation of the affected area.
Mixed type.

52. Laboratory Diagnosis:
 The specimen:
Collected from any ulcerated nodules of the skin or from the nasal secretion as well as by gentile scraping of the nasal septum.
 Direct Examination:
Film stained with modified Ziehl Neelsen stain, the presence of acid and alcohol fast bacilli, usually in groups, intracellular is diagnostic.

53. Immunization: Treatment:  Indirect allergic test or Lepromin test:
It is similar to tuberculin test; however, the results are unreliable.
Immunization:
BCG can also be given to protect against leprosy especially for individuals between years old.
Treatment:
Dapsone in combination with clofazimine and rifampin.

54. Prevention and Control:
No vaccine is available.
Isolation of cases and their treatment for years or until the disappearance of symptoms.
Chemoprophylaxis with dapsone of exposed children.

55. Actinomyces and Nocardia

56. General Features:
They are true bacteria (related to corynebacteria and mycobacteria), but they form long branching filaments that resemble the hyphae of fungi.
They are microaerophilic or anaerobic on primary isolation.
Nocardia are aerobic organism.

57. Produce abscesses with yellow granules
Actinomyces
Nocardia
Staining
characters
Gram-positive
Weakly Gram-positive and acid-fast
Oxygen
requirement
Strict or facultative anaerobes
Strict Aerobes
Epidemiology
Part of normal flora (oropharynx, gastrointestinal tract)
Prevalent in soil; occasionally found in sputum of normal individuals.
Clinical disease
Produce abscesses with yellow granules
(sulfur granules)
Cause subcutaneous and pulmonary infection
Treatment
Penicillin G
Sulfonamides

58. Actinomyces israelii

59. General Features:
Gram-positive bacteria which grow in filaments that readily break up into rods and may show branching.
Non-motile.
Non-spore-forming.
Non-acid fast.

60. Culture characteristics:
In tissue, colonies develop to show the diagnostic yellow "sulfur granules", which is visible by naked eye and is found in pus discharge through draining sinuses
Culture characteristics:
A washed, crushed sulfur granule should be used for culture.
Grow on blood or serum glucose agar anaerobically. Incubation at 37° C for at least 7 days.

61. The growth is enhanced by 5% CO2.
Colonies are small, cream, adherent and nodular.

62. Pathogenesis and Clinical disease:
Actinomyces is endogenous in origin and results in a chronic granulomatous infection with abscess formation.
Profuse pus discharges by draining through sinuses.
Infection probably starts after local trauma, e.g. the extraction of carious teeth, appendectomy.
Intra-utrine contraceptive devices; are often colonized with Actino israelii.

63. The significance of this uncertain but, perhaps in association with other organisms, the organism may cause low-grade intra-utrine infection.
Treatment:
Penicillin, licomycin or tetracyclines.

64. Nocardia asteroids Pathogenesis and Clinical disease:
Generally cause granulomatous suppurative infections.
It affects lungs with secondary brain abscess.
It is opportunistic pathogen and affects usually immunocompromized patients.

65. Nocardia madurae and N. brasiliensis
Affect subcutaneous tissues; “madura foot" or mycetoma which is a tropical form of nocardiosis and affects foot and produces chronic discharging sinuses.
Treatment:
Surgical treatment.
Trimethoprim-sulfamethoxazole.