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Mycobacterium
Important Human Pathogens
Mycobacterium tuberculosis
Mycobacterium leprae (uncommon)
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
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 wellaerated upper lobes of the lungs. M bovis like reduce oxigen
Facultative intracellular parasite, usually of macrophages
Slow generation time, 15-20 hours,
Straight or slightly curved rode (3x0.3 mM)
Single in clinical samples-Serpentine cords in culture
Mycobacterium tuberculosis. Acid-fast stain
NOTE: single growth of virulent strains
Acid-Fast (Kinyoun) Stain of
Mycobacterium
NOTE: cord growth (serpentine
arrangement) of virulent strains
Lipid-Rich Cell Wall of Mycobacterium
Mycolic acids
CMN Group:
Unusual cell wall
lipids (mycolic
acids,etc.)
(Purified Protein Derivative)
Corynebacterium, Mycobacterium, Nocardia
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
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)
Pathogenesis of Tuberculosis
• Inhalation of small (1-5 mm) droplet nuclei
containing M. tuberculosis expelled by coughing,
sneezing, or talking of another individual with
cavitary tuberculosis
• Primary infection by M. tuberculosis of nonimmune alveolar macrophages with unrestrained
proliferation within the infected macrophages
forming initial lesion or Ghon focus
• Primary complex (PC): Hilar lymph nodes with
GF
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
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.
Diagram of
a Granuloma
NOTE: ultimately a
fibrin layer develops
around granuloma
(fibrosis), further
“walling off” the
lesion.
Activated Macrophages
Typical progression in
pulmonary TB
involves caseation,
calcification and
cavity formation.
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
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)
Pathogenesis of Tuberculosis
Post-primary
tuberculomas
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)
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)
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
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
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)
PPD Tuberculosis Skin Test Criteria
PPD = Purified Protein Derivative from M. tuberculosis
Chest X-Ray of Patient with Active
Pulmonary Tuberculosis
Lab Diagnosis
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Specimen
Microscopy
Culture
Nucleic acid
Drug susceptibility test
Treatment
BCG (bacille Calmette-Guerin) = attenuated M. bovis
Mycobacterium leprae
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
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- 1010 bacilli/gm diseased tissue (Skin test reagent –
Leprosin A)
Mice footpads 106 (antileprosy drugs)
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
Pathogenesis
First signes
Non specific skin lesion
Spontanuas healing
Disease progress
Clinical manifestation by the immune response of the
patiant (Table 18:7)
Classification
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.
Erythema nodosum leprosum
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.
Clinical Pathology of Leprosy
Tuberculoid (TT) leprosy
The lesions are scanty, dry, erythematous, hypopigmented 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.
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
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.
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
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.
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 wellpreserved for a long time and slowly become fibrotic.
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.
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.
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.
Leprosy Reaction
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.
Leprosy Reaction
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.
Type 1 leprosy reaction
TTS in Type 1 reaction (RR)
Type 2 reaction (ENL)
Type 2 reaction (ENL)
Type 2 Reaction in LL (ENL
Necroticans)
ENL necroticans improved 2 weeks after
Thalidomide administration
Treatment
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.
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 oilimmersion 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).
Lepromatous vs. Tuberculoid Leprosy
Lepromatous Leprosy (Early/Late Stages)
Lepromatous Leprosy Pre- and
Post-Treatment
Chapter 19
Environmental mycobacteria
Home study
actinomyces nocardia and
tropheryma
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
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
Taxonomy of the Aerobic Actinomycetes:
Pathogenic Genera
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Nocardia
Actinomadura
Streptomyces
Rhodococcus
Gordonia
Tsukamurella
Tropheryma whipplei (Non-cultivable)
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
Anaerobic Actinomyces: Natural
Habitats
Anaerobic Actinomyces species
are normal inhabitants of the
mucous membranes of humans
and animals
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
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
Anaerobic Actinomyces: Modes of
Infection
Actinomyces invades normally
sterile tissue from endogenous
mucous membrane sites of
normal colonization
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
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
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
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
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.
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)
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;61:377-378
Model illustrating the pathophysiology of Whipple's disease. DC, dendritic cells
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.
Aerobic Actinomycetes: Identification
• Nocardia and Rhodococcus (potentially
pathogenic) and Streptomyces (less frequently
pathogenic) obligate aerobes
• Nocardia asteroides complex organisms thin
(0.5-1.0 mm) filaments up to 20 mm in length
demonstrating beaded gram-positivity
• Rhodococcus equi gram-positive coccobacilli
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
Aerobic Actinomycetes: Identification
• Nocardia and Rhodococcus are partially acidfast 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
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