Mycobacterium

Family – Mycobacteriaceae

• Mycobacterium consists of more than 100

different species of organisms
• Majority of them saprophytic
• Three major groups (based on the type of
diseases)
1. Tuberculosis Mycobacteria (Mycobacterium
tuberculosis complex (MTB complex))
–M. tuberculosis (Humans)
–M. bovis
–M. canetti
–M. africanum
–M. microti
–M. pinnipedii,

MTB complex- closely related species to M.

tuberculosis
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2. Mycobacterium leprae- cause leprosy
3. Non-tuberculosis Mycobacteria (Atypical
Mycobaterium), Environmental Mycobacteria
– M. kansasii
– M. avium
– M. intracellularae
– M. scrofulaceum
– M. uclerans
– M. marinum
– M. xenopii
– M. fortuitum
M. avium and M. intracellularae is also called M.avium complex
(MAC, have same biochemical reaction)

4
 Important Human Pathogens

• Mycobacterium tuberculosis

• Mycobacterium leprae

• Mycobacterium avium-intracellulaire
Complex (MAC) or (M. avium)
 General Characteristics
• Slender, slightly curved or straight rod-
shaped organisms
• Non-motile
• Do not form spores
• Strictly aerobic
• do not form capsules, flagella or spores
• possess mycolic acids & a unique type of
peptidoglycan
• grow slowly
• Various species found in the soil and water
General characteristics
All bacteria in these group are acid fast
Contain mycolic acid in their cell wall (Contains 60 to 90
carbons)
Lipid rich cell wall, 60% of the cell wall is (mycolic acids, cord
factor, wax-D)
Cell wall is responsible for:-Acid fastness, Slow growth,
resistance to detergents, resistance to many common
antibiotics, antigenicity etc.,
High lipid contents of the cell wall component gives M.tb its acid
fastness, enabling to retain basic dyes like Carbon fuchsine (after
treatment with acid-alcohol)
Cell wall resembles cell wall of Gram positive bacteria but more
complex
M tuberculosis as causative
agent for tuberculosis

1886
Robert Koch
Etiology
• TB is a chronic disease caused
by MTBc
• MTBc consists of 7 spps

M. tuberculosis
M. bovis
M. africanum
MTBc M. microti
M. canettii
M. caprae
M. pinnipedii
Mycobacterium tuberculosis
• M. tuberculosis is an intracellular pathogen that is able to
establish life long infection.
Virulence factors of M. tuberculosis

• M.tb do not have classical virulence factors like

toxins, enzymes/ adhesins

• Cell wall components

Mycolic acid (Long chain FA, C60-C90)
Trehalose 6, 6' dimycolate (cord factor)
Wax D
 Virulence factor con’d
I. High concentration of cell wall mycolic acids (lipids) a long chain
fatty acid (c60-c90) responsible for
– Impermeability to stains and dyes

– Resistance to many antibiotics

– Resistance to killing by acidic and alkaline compounds

– Resistance to osmotic lysis via complement deposition

– Resistance to lethal oxidations and survival inside of

macrophages
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 Virulence determinants
II. Cord factor: made of Trehalose 6, 6- dimycolate
– Responsible for the cording, toxic to mammalian cells and is also
an inhibitor of PMN cells migration
– Abundantly produced in virulent strains of MTB

III. Mycobacterial sulfolipids

IV. Slow generation time (15 – 20 hour)
– The immune system may not readily recognize the bacteria or
may not be triggered sufficiently to eliminate them.
V. Intra cellular growth/survival
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Epidemiology
• World population ~ 7 billion
• ~ 1in 3 people in world infected
• ~ 9.4 million new cases of active TB/year
• About 2 million deaths/year
• In this country most cases occur in the those infected with HIV,
the urban poor, alcoholics, iv drug users, the homeless, migrant
farm workers, immigrants, and prison inmates
• Disease in the elderly usually represents reactivation of a
previous infection
• Disease in children often represents active transmission within
the community or family (they get it from someone else!)
TB epidemiology in Ethiopia
30 high-burden countries for TB, TB/HIV
and multidrug-resistant TB
WHO estimated that there were 200,000
new TB cases in Ethiopia,
ranks 10th among the world’s 22 high-burden
countries for TB, and 4th in Sub-Saharan Africa
TB kills an estimated of 32,000 Ethiopians
every year (more than 80 people per day)
TB is the 3rd cause of hospital admission
and the 2nd cause of death
Has exceptionally highest rates of EPTB
– >80% of EPTB is lymph node TB
Transmission
Transmission
of
of M.tb
M.tb
 How is TB Transmitted?
• Person-to-person through
the air by a person with TB
disease of the lungs
• Spread by
• coughing,
• sneezing,
• Singing or
• talking
Source: CDC, 2000

 Less frequently transmitted by:

• Ingestion of Mycobacterium bovis found
in unpasteurized milk products
• Laboratory accident
 Transmission of M. tuberculosis
 Millions of tubercle bacilli in
lungs (mainly in cavities)
 Coughing projects droplet nuclei
into the air that contain tubercle
bacilli

• One cough can release 3,000

droplet nuclei

• One sneeze can release tens of

thousands of droplet nuclei
 The Chance of Infection Increases…
• When the concentration of TB bacteria circulating in the
air is greater
– Coughing; smear +; cavitary disease
– Exposure occurs indoors
– Poor air circulation and ventilation; small, enclosed
space
– Poor or no access to sunlight (UV light)
– The greater the time spent with the infectious person
or breathing in air with infectious particles
TB Germs Cannot be Spread By:

• Sharing dishes and utensils

• Using towels and linens
• Handling food
• Sharing cell phones
• Touching computer keyboard
 TB Transmission and Pathogenesis (3)

No infection (70%)
Adequate

Exposure Non-immunologic Early progression (5%)

defense
Inadequate
Inadequate
Immunologic
Infection (30%) defenses Late progression(5%)
Inadequate
Adequate
Containment (95%) Immunologic
defenses
Adequate
Continued containment (90%)
Person:
 Not ill
 Not contagious
 Normal chest x-ray
 Usually the tuberculin
skin test is positive
Germs:
 Sleeping but still alive--?
 Surrounded (walled off)
by body’s immune
system
 Active TB Disease
TB
Germs:
• Awake and multiplying
• Cause damage to the lungs
Person:
• Most often feels sick
• Contagious (before pills started)
• Usually have a positive tuberculin
skin test
Granuloma breaks • Chest X-ray is often abnormal
down and tubercle (with pulmonary TB)
escape and multiply
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
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cavity formation.
 Primary TB
• infectious dose 10 cells
• phagocytosed by alveolar macrophages
& multiply intracellularly
• after 3-4 weeks immune system attacks,
forming tubercles, granulomas
consisting of a central core containing
bacilli surrounded by WBCs
• If the organism does not cause
immediate infection, the organism can
be “walled off” in a granuloma
• Granulomas can break down in future
and the organisms can cause infection
later
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 High Risk for Progression
Persons more likely to progress from LTBI to TB
disease include:
Persons with certain medical
conditions such as:
• HIV-infected persons  Diabetes mellitus
• Persons with a history of  Chronic renal failure or on
prior, untreated TB or hemodialysis
fibrotic lesions on chest X-
ray  Solid organ transplantation
 Certain types of cancer (e.g.,
• Recent TB infection (within
past 2 years) leukemia)
 Gastrectomy or jejunoileal
• Injection drug users
bypass
• Age (very young or very  Underweight or malnourished
old) persons
 Silicosis
Spread of TB to Other Parts of the Body

1. Lungs (85% all cases)

2. Pleura
3. Central nervous system
• (e.g., brain, meninges)

4. Lymph nodes
5. Genitourinary system
6. Bones and joints
7. Disseminated
– (e.g., miliary)
 TB Can Affect Any Part of Your Body:
Extrapulmonary TB

• Extrapulmonary
tuberculosis
– Lymph node
– Spleen
Brain – Liver
Pleura
– Lungs
– Bone marrow
– Kidney
– Adrenal gland

Lymph Node Spine

MTBc Pathogenesis
TB Pathogenesis
• Bacterial entry
• T Lymphocytes.
• Macrophages.
• Epitheloid cells.
• Proliferation.
• Central Necrosis.
• Giant cell
formation.
• Fibrosis.
 Morphology of Granuloma
1. Rounded tight collection of chronic inflammatory
cells.

2. Central Caseous necrosis.

3. Active macrophages - epithelioid cells.
4. Outer layer of lymphocytes & fibroblasts.
5. Langhans giant cells – joined epithelioid cells.
 Secondary tuberculosis
• Definition: the infection of an individual who has been
previously infected or sensitized
• The infection may be acquired from
– Endogenous source: reactivation of dormant primary complex
– Exogenous source
Fate of secondary tuberculosis
•The lesion may heal with fibrous scarring and calcification
•Fibrocaseous tuberculosis (progressive pulmonary TB )
•Can progress to EPTB- Extrapulmonary (disseminated) TB,
bacilli disseminate to regional lymph nodes, kidneys, long
bones, genital tract, brain, meninges = Bad News
•Miliary tuberculosis: Occurs when organisms drain through
lymphatics into lymphatic ducts
Secondary Tuberculosis

Figure 19.22b
Evaluation for TB
• Medical history
• Physical examination
• Mantoux tuberculin skin test/ IGRAs
• Chest x-ray
•Histopathology/cytology
• Bacteriologic exam
• smear
• Culture
• NAAT/PCR 34
 Symptoms of TB
• Productive prolonged cough( >2wks)*
• Chest pain*
• Hemoptysis*
• Fever and chills
• Night sweats
• Fatigue
• Loss of appetite
• Weight loss
Chest x-Ray
• Obtain chest x-ray for patients with positive TST results or with
symptoms suggestive of TB
• Abnormal chest x-ray, by itself, cannot confirm the diagnosis of TB
but can be used in conjunction with other diagnostic indicators

The most common abnormality

associated with primary TB on
chest radiography is
•hilar adenopathy
•Subpleural granulomas
 Laprotary Diagnosis of Tuberculosis
Specimens: Sputum, Urine, pleural fluid, peritoneal fluid, C.S.F.,
gastric lavage, lymph node biopsy, tissue biopsy
Microscopic Examination:
1. Light microscopy; staining by Z.N
• Specific & rapid
• Cheap
• Low sensitivity (65 - 70% with repeated smear) ----- HIV??
• Limit of detection is 5000-10000bacilli/ml of sputum
2. Fluorescence microscopy.
• Using auramine staining
• 10% more sensitive than ZN
• Allow rapid examination
3. Cytology → using FNA / biopsy samples
AFB - Ziehl-Nielson stain
Microscopic Features- cytology

Granulomas with central

caseation

Epithelioid granulomas with

Langhans giant cells
typically associated with TB
 Culture
• Mycobacterium are strictly Media- 3 types
aerobic -Egg-Based with
Malachite green (inhibits
• 5-10% CO2 bacteria)
• 35-37oC Lowenstein-Jensen (LJ)
• Slow growers; cultures -Requires about 3 - 8 weeks
held for 6 weeks before for growth to occur
calling negative
-Agar based
• Used to confirm diagnosis of Promotes early growth
TB--- Gold standard method— Middlebrook 7H10 and
• More sensitive than smear 7H11 agar – serum
based
microscopy
• Culture all specimens, even if -Liquid Media
smear is negative Middlebrook 7H9 Broth
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ID of M. tuberculosis --CULTURE

• Slow grower
• Colonies are thin, flat,
spreading and friable with
a rough appearance
• May exhibit characteristic
“cord” formation
• Grows best at 35 to 37° C
• Colonies are NOT
photoreactive
Colony Morphology – LJ Slant
4. Molecular Techniques
– Allow rapid detection of MTBc DNA
– Routine PCR: Gene amplification
– Real Time PCR: Gene amplification plus quantification
– Sensitivity: 80 - 85%, specificity ~ 99%
• GeneXpert MTB/RIF assay
– Is the novel diagnostic tool endorsed by WHO in 2012---
– Detects MTBc and its resistance to rifampicin
• Can also detect mutations coffering drug resistance
Mantoux test (TST) Intracutaneous injection of
Tests for exposure-- LTBI purified protein derivative (PPD)
Tests for hypersensitivity(type IV) Induration measured after 48-72h

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PPD result after
– 72 hours
 Treatment of Latent TB Infection
• Daily Isoniazid therapy for 9 months
– Monitor patients for signs and symptoms of
hepatitis and peripheral neuropathy
• Alternate regimen – Rifampin for 4 months

BCG vaccination
• vaccine based on attenuated bacilli Calmet-
Guerin (BCG) strain of M. bovis

47
 Treatment of active TB Disease
• Include four 1st-line drugs in initial regimen
– Isoniazid (INH)
– Rifampin (RIF)
– Pyrazinamide (PZA)
– Ethambutol (EMB)
• Adjust regimen when drug susceptibility results
become available or if patient has difficulty with any of
the medications
• Never add a single drug to a failing regimen
• Promote adherence and ensure treatment completion
 Directly Observed Therapy (DOT)
• Health care worker watches
patient swallow each dose of
medication
• DOT is the best way to ensure
adherence
• Should be used with all
intermittent regimens
• Reduces relapse of TB disease
and acquired drug resistance

49
 Clinical Monitoring
Instruct patients taking TB medications to
immediately report the following:
– Rash
– Nausea, loss of appetite, vomiting, abdominal pain
– Persistently dark urine
– Fatigue or weakness
– Persistent numbness in hands or feet

50
Treatment of Multidrug resistant TB
• MDR-TB, a MTBc strain resistant to RIF and INH
• Multi-drug resistant TB treatment: much more expensive,
toxic and can go on for 2 years.
• Mycobacterium is fairly resistant and only a few
organisms left can cause reinfection
• Development of drug-resistance
– Inadequate treatment regimes
– Patient noncompliance
– Mutations
• Second line anti-TB drugs: Kanamycine, Capreomycin
Cycloserine, Ethionamide, Ciprofloxacin
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Antibiotic Sensitivity Testing
• Culture –phenotypic DST
– Solid media
– Liquid media
• Molecular techniques
– Line probe assay
• Detection of resistance to RIF
& INH
– GeneXpert MTB/RIF
• Detection of resistance to RIF
Prevention & control of TB
– Early case detection & treatment

– Decreasing of over crowding

– Pasteurization of milk --- ↓ M. bovis infection

– Immunization (BCG)

– Health education
 Mycobacterium bovis
• Morphologically similar to M.tb
• Is primary a zoonotic diseases.. Bovine TB
• Primarily in cattle, dogs, cats, swine, parrots and
human; disease in humans
– Slow grower
– Small, granular, rounded white colonies with
irregular margins
– Nonpigmented
– Similar to M. tuberculosis
Armauer Hansen in 1868 55
 Mycobacterium leprae
• Gram-positive,
• Aerobic rod-shaped bacillus
• Intracellular
• With a waxy coating
• M. leprae is unable to grow in vitro
• strict parasite – has not been grown on artificial media/tissue culture
• slowest growing of all species
• causes leprosy, a chronic disease that begins in the skin & mucous
membranes & progresses into nerves
• It has a predilection for cooler areas of human body (skin, peripheral
nerves, nose, anterior chamber of the eyes).
• The skin and nerves are the most commonly affected organs. 56
57
Armadillo

Some types of monkeys

Rabbits and Mices (foot pads)

 Leprosy is a chronic granulomatous
diseases caused by Mycobacterium
Leprae.
 It is also known as Hansen disease
 It is regarded as public health
problem
 permanent disabilities
 social consequences of
discrimination and stigma
 Leprosy
M. leprae is transmitted from human to
human through
– prolonged contact; for example, between
exudates of a leprosy patient's skin
lesions and the abraded skin of another
individual and
– more commonly through nasal droplets
from a patient with lepromatous disease.

•Source : Nasal or Skin

– skin -Inoculation.
– Nasal mucosa- Inhalation
Leprosy

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Hypopigmented Nerve enlargment
patches
Nodules on the ears

Neurological deficits
 64
Leprosy
Leprosy

66
Pathogenicity:
@ Organism multiplies inside nerve cells,
endothelial cells, and skin histiocytes.

@ Two forms of leprosy:

# Tuberculoid leprosy
 Skin macules, hypopigmentation,
skin thickening, skin anaesthesia.

# Lepromatous leprosy
 Skin nodules, leonine facies, and
disfigurement.
 Immunology
Tuberculoid leprosy
-Patient’s lymphocytes respond to M. leprae in vitro
-Skin tests with lepromin elicit a strong positive response
-They also have a Th1- type response producing interleukin-2 and
interferons-γ
-These strong cell-mediated responses clear antigens, but cause local tissue
destruction
Lepromatous leprosy
-Patients in this case do not mount a normal cell mediated response to M.
leprae, and in fact their lymphocytes do not respond to M. leprae in vitro
-They are also unresponsive to lepromin
-They have specific T cell failure and macrophage dysfunction, and problems
producing interleukin-2 and interferons-γ
-But they do produce Th2-type cytokines

68
Leprosy

69
Lepromatous leprosy Lepromatous leprosy

70
The major complication
-Peripheral neuropathy.
71
Differences between Tuberculoid & Lepromatous
leprosy
Tuberculo
Feature Lepromatous
id

Number of Few Many

lesions

Tissue Little Marked

destruction
Few Many
Bacilli in smears
Low High
Transmission
Reduced or
Cell-mediated Present
absent
reaction
Specimen:
•Ear lobe scrapings, nasal scrapings, eye Clinical findings
brow scrapings, tissue biopsy, nerve •Lesions involving:
biopsy •Skin (causing
Ziehl Neelson staining technique disfigurement)
 Look for bacilli (at least examine 200 •Superficial nerves
fields) •Nose, pharynx, eyes and
Laboratory diagnosis of lepromatous testicles
leprosy, where organisms are numerous, – Lymphadenopathy
involves acid-fast stains of specimens •Incubation period- 2 - 10
from nasal mucosa or other infected years
areas.
In tuberculoid leprosy, organisms are
extremely rare.
Acid-fast stains
Lepromin Test:
•Skin test for delayed hypersensitivity to
leprae bacilli.
•Indicates cell-mediated
reaction
•Antigens: Boiled extract of Lepromatous
tissue in isotonic saline.
@ Inject Myco. leprae extract
I.D.

@ Induration appears within 48

hrs.

@ Lepromin test is diagnostic if

negative.
Treatment
– Dapsone & rifampin
– Suppress the growth of M. leprae
N.B: Without prophylaxis, about 10% of exposed children acquire
the disease
• WHO recommended Multi drug therapy

• Prevention and control

– Identification & treatment of cases
– Provision of chemopropylaxis------ risk groups
– BCG vaccination -------endemic areas
– Health education
 NTM (Nontuberculous
mycobacteria)

ATYPICAL MYCOBACTERIA

77
 Other Mycobacteria
• MOTT (Mycobacteria Other Than Tubercle
Bacillus)
• NTM (Nontuberculous mycobacteria)
• >50 species
• Saprophytes, mostly from water and soil
• Animal pathogens
• Opportunistic pathogens in humans
• Person-person transmission rare
 ATYPICAL MYCOBACTERIA
Classification:
Group I (Photochromogens):
slow growing, produce yellow-orange
pigment in light only.
Group II (Scotochromogens): Slow growing,
produce yellow-orange pigment in light and
darkness.
Group III (Nonchromogens): Slow growing,
produce no pigment.
Group IV (Rapid growers): Grow within 2-3 days &
produce no pigment.
 Other Mycobacteria
(cont’d)
• NTM
– Photochromogens
• M. kansasii
• M. marinum
– Scotochromogens
• M. gordonae
• M. scrofulaceum
– Nonphotochromogens
• M. avium Complex (MAC)
– Rapid Growers
• Mycobacterium fortiutum-chelonei Complex
Diseases of Atypical Mycobacteria:
84
Any questions ???