1. Mycobacterium

2. Important Human Pathogens
Mycobacterium tuberculosis
Mycobacterium leprae (uncommon)

3. Mycobacterium tuberculosis complex
M. tuberculosis: Human TB
M. caprae: Goat, Vet surgeons
M. bovix: cattle and other mammals
M. microti: Voles and other small animals/ not human
M. africanum: Africa in btw Human and bovine types

5. Mycobacterium Description
M. Tuberculosis complex (tubercle bacilli)
Non-motile, non-sporing, non-capsulate.
Obligate aerobe: For this reason, in the classic case of tuberculosis, MTB complexes are always found in the well-aerated upper lobes of the lungs. M bovis like reduce oxigen
Facultative intracellular parasite, usually of macrophages
Slow generation time, hours,
Straight or slightly curved rode (3x0.3 mM)
Single in clinical samples-Serpentine cords in culture

6. Mycobacterium tuberculosis. Acid-fast stain
NOTE: single growth of virulent strains

7. Acid-Fast (Kinyoun) Stain of Mycobacterium
NOTE: cord growth (serpentine arrangement) of virulent strains

8. Lipid-Rich Cell Wall of Mycobacterium
Mycolic acids
CMN Group: Unusual cell wall lipids (mycolic acids,etc.)
(Purified Protein Derivative)
Corynebacterium, Mycobacterium, Nocardia

9. Mycobacterium Description
Grow in wide range of enriched media
the common for clinical isolation.
Löwenstein-Jensen (LJ) medium is an egg based medium
Middlebrook's medium (MTB) is an agar based medium

10. Photochromogenic Mycobacterium kansasii on Middlebrook Agar
NOTE: Mycobacteria pathogenic for humans can be differentiated (Runyon Groups) by:
speed of growth (all are slower than most other pathogens) and by
production of chromogenic pigments (in light, in dark, or none)

11. Pathogenesis of Tuberculosis
Inhalation of small (1-5 m) droplet nuclei containing M. tuberculosis expelled by coughing, sneezing, or talking of another individual with cavitary tuberculosis
Primary infection by M. tuberculosis of non-immune alveolar macrophages with unrestrained proliferation within the infected macrophages forming initial lesion or Ghon focus
Primary complex (PC): Hilar lymph nodes with GF

12. Pathogenesis of Tuberculosis
H. bovis PC formed in the tosil, cervical nodes or ileocaecal region and mesentric lymph nodes.
Prosector’s warts: primary focus on the skin

13. Pathogenesis of Tuberculosis
Dissemination of infected macrophages through the draining lymphatics into the circulation
Development within 3-8 weeks of a CD4+ T cell dependent cell-mediated immune response with granuloma formation and macrophage activation at sites of infection by IF- and calciriol.

14. 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.
Activated Macrophages

15. Immunity in Tuberculosis
Antigen-specific activation of CD4+ T lymphocytes with secretion of IL-2, increased expression of IL-2 receptors, and secretion of IF-
Antigen-driven clonal expansion of CD4+ T lymphocytes by IL-2 acting via autocrine and paracrine mechanisms
Activation by IF- of Mycobacterium tuberculosis killing by macrophages

16. Pathogenesis of Tuberculosis
Minority of cases Foci progresses to
Progressive primary lesions, meningitis, pleurisy and kidney, spine (pott’s disease), bone, joint)
Miliary tuberculosis: focus ruptures into blood
(table 18.2)

17. Pathogenesis of Tuberculosis

18. Post-primary
tuberculomas

19. Pathogenesis of Tuberculosis
Active infection usually transformed into latent infection (exceptions: infants, AIDS)
With decrement in T-cell dependent cell mediated immunity (years later) infection reactivated with development of tuberculosis (HIV infection, diabetes mellitus, renal disease, cancer, advanced age)

20. Pathogenesis of Tuberculosis
Reactivation of M. tuberculosis infection with partial immunity produces high tissue concentrations of mycobacterial antigens that provoke an intense mononuclear cell response (type 4 hyper- sensitivity reaction)

21. Pathogenesis of Tuberculosis
Dense mononuclear cell infiltrates damage tissue due to release of active oxygen radicals and lysosomal neutral proteases
Tissue damage occurs as caseation necrosis that progresses to liquefaction necrosis in the absence of tuberculosis drug treatment

22. Clinical Features of Tuberculosis
Cough one of the earliest signs with production of sputum as tissue necrosis progresses
Dyspnea a later symptom indicating extensive involvement of pulmonary parenchyma
Fever and weight loss reflecting systemic actions of IL-1 and TNF- (cachectin) secreted by activated macrophages

24. Clinical Features of Tuberculosis
Apical cavitary lesions in upper lobes of lung by X-ray film of the chest
Positive tuberculin skin test with PPD (purified protein derivative)

25. PPD Tuberculosis Skin Test Criteria
PPD = Purified Protein Derivative from M. tuberculosis

26. Chest X-Ray of Patient with Active Pulmonary Tuberculosis

27. Lab Diagnosis Specimen Microscopy Culture Nucleic acid
Drug susceptibility test

30. Treatment
BCG (bacille Calmette-Guerin) = attenuated M. bovis

31. Mycobacterium leprae

32. Mycobacterium leprae
Mycobacterium leprae is the causative agent of the disease, leprosy (Hanson's Disease).
gram-positive, aerobic rod , not so strongly Acid fast
The bacilli are typically found within Mf in clumps.
Surface lipid, peptidoglycolipid1 (PGL-1) is a unique carbohydrate antigenic determinant.
PCR specific primers

33. Mycobacterium leprae
M. leprae has never been grown in artificial culture, but will grow in the footpads of mice and in armadillos. The culture can take several weeks to mature.
Armadillos bacilli/gm diseased tissue (Skin test reagent –Leprosin A)
Mice footpads 106 (antileprosy drugs)

35. Pathogenesis Principal target cells is Schwann cells Nerve damage
anaesthesia and muscle paralysis
Repeat injuries and infection
Gradual destruction
Infiltration of skin & cutaneous nerves
Visible lesion wth pigmentary changes

36. Pathogenesis First signes Non specific skin lesion Spontanuas healing
Disease progress
Clinical manifestation by the immune response of the patiant (Table 18:7)

37. Classification BT (borderline tuberculoid)
The dichotomy: multibacillary or paucibacillary
The Ridley system:
TT (polar tuberculoid)
BT (borderline tuberculoid)
BB (borderline/midborderline)
BL (borderline lepromatous)
LLs (subpolar lepromatous)
LLp (polar lepromatous)
The paucibacillary: TT and most BT.
The multibacillary: BB, BL, LLs and LLp.
Lepromin skin test: classification of diagnosed patients
Positive: all TT and most BT.
Negative: others.

38. Erythema nodosum leprosum

39. Clinical Pathology of Leprosy
Early, Indeterminate Leprosy
Slight pandermal perineurovascular and peri-appendageal chronic inflammation.
Without demonstrating bacilli:
Diagnosis can only be presumptive
Lepromatous leprosy (LL)
The lesions usually are numerous and symmetrically arranged.
Three clinical types:
macular, infiltrative-nodular and diffuse.
A distinctive variant of LL: histoid type.

40. Clinical Pathology of Leprosy
Tuberculoid (TT) leprosy
The lesions are scanty, dry, erythematous, hypo-
pigmented papules or plaques with sharply defined
edges.
Anesthesia is prominent (except on the face).
The number of lesions ranges from 1 to 5, and the
lesions heal rapidly on chemotherapy.
Primary TT leprosy has large epitheloid cells arranged
in compact granulomas along with neurovascular
bundles with dense peripheral lymphocyte accumulation
Langhans’ giant cells are typically absent.
Dermal nerve may be absent or surrounded and eroded
by dense lymphocyte cuffs. Bacilli are rarely found.

41. Clinical Pathology of Leprosy
Borderline tuberculoid (BT) leprosy
The lesions are asymmetrical and may be scanty.
Dry, hairless plaques with central hypopigmentation.
Nerve enlargement is usually found and the lesions
are usually anesthetic.
Granulomas with peripheral lymphocytes follow the
neurovascular bundles and infiltrate sweat glands and
erector pili muscles.
Langhans’ giant cell are variable in number and are
not large in size.
Typical nerve erosion and destruction.
Granulomas along superficial vascular plexus frequently

42. Clinical Pathology of Leprosy
Midborderline (BB) leprosy
The lesions are irregularly dispersed and shaped
erythematous plaques with punched-out centers.
Dermal edema is prominent in the lesions.
Macrophages are activated to epitheloid cells
Borderline lepromatous (BL) leprosy
Less numerous and less symmetrical.
Often display some central dimples.
The lymphocytes are more prominent in BL and
there is a tendency for some activation of macrophages
to form poorly to moderately defined granulomas.
Perineural fibroblast proliferation forming an typical
“onion skin” in cross section.

43. Clinical Pathology of Leprosy
Infiltrative-nodular type LL
The classical and most common variety.
The patients are not notably hypoesthetic disturbances of sensation and nerve paralyses develop after large peripheral nerve involved.
Most common involved nerve:
ulnar, radial and common peroneal nerves.
Extensive cellular infiltrate with separated from the
epidermis by a narrow grenz zone of normal collagen.
The macrophages have abundant eosinophilic cytoplasm
and contain mixed solid and fragmented bacilli .
Lymphocyte infiltration is not prominent, but there may
be many plasma cells

44. Clinical Pathology of Leprosy
Hitoid type LL
The occurrence of well-demarcated cutaneous and
subcutaneous nodules resembling dermatofibromas.
It frequently follows incomplete chemotherapy or
acquired drug resistance, leading to bacterial relapse.
The highest loads of bacilli.
The majority are solid-staining, arranged in clumps
like sheaves of wheat.
Macrophage reaction is unusual.

45. Clinical Pathology of Leprosy
Lepromatous leprosy -- with antimycobacterial therapy
Degenerate bacilli accumulate in the macrophages
(the so-called lepra cells or Virchow cells), which
have foamy or vacuolated cytoplasm, resembling
xanthoma cells
Fite stain reveals that the bacilli are fragmented or
granular and disposed in large basophilic clumps
called globi especially in very chronic lesions.
The nerves in the skin may contain considerable
numbers of leprosy bacilli, but remain well-
preserved for a long time and slowly become fibrotic.

46. Clinical Pathology of Leprosy
Lepromatous leprosy -- with antimycobacterial therapy
When lepromatous leprosy is treated, the bacterial
debris to be cleared by host macrophages.
The M lepra antigen may persist longer and can be
demonstrated by immunocytochemical stains even when no bacilli are evident.

47. (Jopling’s type 1 reaction) CMI
Leprosy Reaction
The reactional status of leprosy are distinctive, tissue destructive, inflammatory processes, putatively immunologically driven greatly increasing the morbidity of the disease.
Delay-Type Hypersensitivity Reaction
(Jopling’s type 1 reaction) CMI
Type 1 reactions are common in TT, BB and BL patients, but are not rare in LL or BL patients.
Reaction induces increased intraneural inflammation
and edema, which is damaging.
At worst, there is a caseous necrosis of large peripheral
nerves resulting from upgrading reactions.

48. Leprosy Reaction
Delay-Type Hypersensitivity Reaction
Edema within and about the granulomas and proliferation of fibrocytes in the dermis.
In upgrading reactions, the granuloma becomes more
epitheloid and Langhans’ giant cells are larger.
There may be erosion of granulomas into the lower
epidermis and fibrinoid necrosis with granulomas and
even within dermal nerves.
In downgrading reactions, necrosis is much less
common and the density of bacilli increases.

49. Leprosy Reaction Ag-Ab complex
Type 2 reaction (Erythema nodosum leprosum, ENL)
Ag-Ab complex
ENL occurs most commonly in LL, less in BL.
Tender, red plaques and nodules together with areas of
erythema and occasionally also purpura and vesicles.
It is accompanied by fever, malaise, arthralgia and
leukocytosis.
This is the only type of reactional leprosy that responds
to treatment with thalidomide.
Polymorph neutrophils may be scanty or so abundant as
to form a dermal abscess with ulceration
Foamy macrophage containing fragmented bacilli or
mycobacterial debris.
A necrotising vasculitis in some cases of ENL.

50. Leprosy Reaction Occur exclusively in diffuse lepromatous leprosy.
Lucio Reaction
Occur exclusively in diffuse lepromatous leprosy.
It usually occurs in patients who have received either
no treatment or inadequate treatment.
The lesions consist of barely palpable, hemorrhagic,
sharply marginated, irregular plaques.
There may be repeated attacks or continuous
appearance of new lesions for years.
Endothelial proliferation leading to luminal obliteration
with thrombosis in the medium-sized vessels of the
dermis and subcutis.
Dense aggregates of bacilli are found in the walls and
the endothelium of vessels.

51. Type 1 leprosy reaction

52. TTS in Type 1 reaction (RR)

53. Type 2 reaction (ENL)

54. Type 2 reaction (ENL)

55. Type 2 Reaction in LL (ENL Necroticans)

56. ENL necroticans improved 2 weeks after Thalidomide administration

57. Treatment WHO: the combination of dapsone (bacteriostatic)
Paucibacillary disease (TT or BT)
WHO: the combination of dapsone (bacteriostatic)
100mg QD and rifampin (bactericidal) 600mg monthly for a duration of 6 months.
Multibacillary disease (BB, BL, and LL)
WHO: dapsone 100mg QD, rifampin 600 mg monthly
and clofazimine (bacteriostatic) 50mg QD and 300mg
monthly for a routine duration of 2 years.
20% relapse rate within 8 years after completion of
this regimen.

58. Identification and Quantitation of Bacilli
 Acid-fast stain: weak.
 Modifications of the Ziehl-Neelsen method
(collectively called Fite-Farraco stains)
 Bacilli are usually found in macrophage and nerves.
 Bacillary index (BI): the numbers of bacilli per oil-
immersion field (OIF) or OIFs sought to find one
bacillus
Other Methods of Diagnosis
 Antibodies directly against phenolic glycolipid I or
lipoarabinomannan.
 Polymerase chain reaction (PCR).

59. Lepromatous vs. Tuberculoid Leprosy

60. Lepromatous Leprosy (Early/Late Stages)

61. Lepromatous Leprosy Pre- and Post-Treatment

62. Environmental mycobacteria
Chapter 19
Environmental mycobacteria
Home study

63. actinomyces nocardia and tropheryma

64. Description of aerobic Actinomycets
Gram-positive branching filaments that sporulate or fragment: the aerobic
Actinomycetes (order Actinomycetales)
Aerobic Actinomycetes whose cell walls contain mycolic acid: Nocardia species and Rhodococcus species (family Nocardiaceae)
Aerobic Actinomycetes whose cell walls lack mycolic acid: Streptomyces species

65. Description of the Anaerobic Actinomyces
Anaerobic non-sporulating gram-positive rods consist of two groups based on guanosine (G) plus cytosine (C) DNA content: Low mole percent (30-53%) and high mole percent (49-68%)
Actinomyces species member of the high G+C group

66. Taxonomy of the Aerobic Actinomycetes: Pathogenic Genera
Nocardia
Actinomadura
Streptomyces
Rhodococcus
Gordonia
Tsukamurella
Tropheryma whipplei (Non-cultivable)

67. Aerobic Actinomycetes: Natural Habitats
Nocardia species and other aerobic
Actinomycetes ubiquitous in soil and primarily responsible for decomposition of organic plant matter
Rhodococcus species present in the intestinal bacterial flora of grazing herbivores especially horses
Streptomyces species (>3,000) widely distributed in soil

68. Anaerobic Actinomyces: Natural Habitats
Anaerobic Actinomyces species
are normal inhabitants of the
mucous membranes of humans
and animals

69. Aerobic Actinomycetes: Modes of Infection
Nocardia infection acquired by inhalation of or direct skin inoculation (traumatic) by environmental organisms
Rhodococcus infection due primarily to inhalation of organisms by animal handlers (horses, pigs, cattle)
Streptomyces are soil organisms that can infect traumatic wounds especially of the feet

70. Aerobic Actinomycetes: Modes of Infection
Actinomadura species (A. madurae, A. latina, A. pelletieri) produce subcutaneous infections in tropical and subtropical countries with those who walk barefooted
Gordonia and Tsukamurella species are closely related to Rhodococcus, and are soil organisms considered opportunistic pathogens

71. Anaerobic Actinomyces: Modes of Infection
Actinomyces invades normally
sterile tissue from endogenous
mucous membrane sites of
normal colonization

72. Aerobic Actinomycetes: Types of Infectious Disease
Nocardia a facultative intracellular parasite that infects human macrophages and inhibits the fusion of phagosomes containing organisms with lysosomes.
Nocardia infections generally occur in immunocompromised patients or those with underlying pulmonary disease

73. Aerobic Actinomycetes: Types of Infectious Disease
Nocardia asteroides complex: N. asteroides sensu stricto type VI, N. abscessus, N. farcinica, and N. nova, major cause of pulmonary infection
N. otitidiscavarium infrequent cause of systemic infection
N. brasiliensis inoculated into subcutaneous tissue of foot produces actinomycotic mycetomas

74. Aerobic Actinomycetes: Types of Infectious Disease
Nocardial pneumonia occurs primarily in immunocompromised hosts and produces necrotizing pyogranuloma formation.
Extrapulmonary dissemination (~50%) and metastatic brain abscess (~30%) complications of nocardial pneumonia.
Actinomycotic mycetoma (pyogenic subcutaneous infection) causes local tissue destruction including bone

75. Aerobic Actinomycetes: Types of Infectious Disease
Rhodococcus equi infects macrophages inhibiting phagolysosome fusion, and produces pulmonary disease with cavitation. Infection occurs in immunocompromised (especially HIV-infected) individuals who handle horses.
R. equi disseminates to other organs including the brain and subcutaneous tissue

76. Aerobic Actinomycetes: Types of Infectious Disease
Streptomyces (S. anulatus formerly S. griseus, and S. somaliensis) associated with actinomycotic mycetoma in warm climates.
S. somaliensis a frequent cause of actinomycotic mycetomas of the head and neck.

77. Aerobic Actinomycetes: Types of Infectious Disease
Whipple’s disease: diarrhea, weight loss, lymphadenopathy, fever, and arthralgia
Typical histopathology is presence of PAS-positive foamy macrophages infiltrating the lamina propria of the small intestine
Caused by intracellular infection of macrophages by Tropheryma whipplei (non-cultivable, diagnosis by typical histopathology combined with PCR)

78. Periodic acid-Schiff (PAS)
Staining method used to detect high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), in tissues. The reaction of periodic acid selectively oxidizes the glucose residues, creates aldehydes that react with the Schiff reagent and creates a purple-magenta color. A suitable basic stain is often used as a counter stain.
Várvölgyi C et al. Ann Rheum Dis 2002;61:

79. Model illustrating the pathophysiology of Whipple's disease
Model illustrating the pathophysiology of Whipple's disease. DC, dendritic cells

80. Anaerobic Actinomyces: Types of Infectious Disease
Actinomyces israelii causes actinomycosis in which chronic granulomas become suppurative. Cervicofacial actinomycosis most common (~60%), followed by abdominal (20%) and pulmonary (15%).
Tissue pus contains sulfur granules, a tangled mass of branching bacteria. Presence of sulfur granules establishes a diagnosis of actinomycosis.

81. Aerobic Actinomycetes: Identification
Nocardia and Rhodococcus (potentially pathogenic) and Streptomyces (less frequently pathogenic) obligate aerobes
Nocardia asteroides complex organisms thin ( m) filaments up to 20 m in length demonstrating beaded gram-positivity
Rhodococcus equi gram-positive coccobacilli

82. Aerobic Actinomycetes: Identification
Nocardia grows in a variety of media including blood and chocolate agars, Sabouraud’s dextrose agar without chloramphenicol, Lowenstein-Jensen slant, Middlebrook agar, and thioglycolate or trypticase soy broth.
Growth is slow requiring 5-7 days up to 3 weeks for colony formation at 25o to 37oC.
Growth in culture of Actinomadura and Streptomyces similar to Nocardia

84. Aerobic Actinomycetes: Identification
Nocardia and Rhodococcus are partially acid-fast positive by modified Kinyoun stain (1% H2SO4 used as decolorizing agent)
Resistance or sensitivity of growth in glycerol broth to lysozyme
Urease activity
Decomposition of the substrates casein, tyrosine, xanthine, and hypoxanthine

86. Anaerobic Actinomyces: Identification
Actinomyces israelii anaerobic with clinical strains varying from obligate anaerobes to microaerophilic
A. israelii definitively identified by detection using gas liquid chromato- graphy (GLC) of acetic and lactic acid as end products of carbohydrate metabolism