Mycobacteriaceae
Aerobic Gram-Positive Bacilli
Form Filaments
Stain Acid-fast
FATHER
DAMIEN
(1840 – 1889)
Lived and died
for victims of
Hansen’s disease
(leprosy)
on Molokai,
Hawaii
Mycobacterium: Genera
• “fungus” “small rod”
• Aerobic, fastidious, slow growing
– Cell division 12-24 hr.
– Lab culture 1-2 months
• Large lipid content in cell wall, resistant to
–
–
–
–
Disinfectants
Detergents
Common antibiotics
Lab basic stains
• ~100 species, many isolated in humans
• Important human pathogens:
– M. tuberculosis – “small swelling”
– M. avian-intracellulare – “birds”; TB-like illness, common in
AIDS patients
– M. leprae – “scaly skin”; Hansen’s disease
Nontuberculosis Mycobacteria:
Runyon Classification
• Based on growth rate, pigmentation
• Non-Runyon Group: M. tuberculosis, M. leprae
• Group I: Slow-Growing Photochromagen
– No pigment grown in dark
– Photoactivated pigment upon exposure to light
• Group II: Slow-Growing Scotochromogen
– Yellow, orange pigments grown in light or dark
– Pigment deepens in two weeks
• Group III: Slow-Growing Nonphotochromagen
– Produce white, yellow pigment
– Pigment not intensify upon exposure to light
– M. avian complex
• Group IV: Rapid Grower
– Colonies in seven days
Mycobacterium: Staining
• G(+) bacilli, slender, branched filaments;
stain poorly because of lipids (mycolic
acid, waxD) in cell wall
• Acid-fast stain
– Presumptive diagnosis mycobacterial disease
– Heating for stain penetration of high lipid
cell wall
– Stain penetrates, binds to mycolic acid, not
remove with acid-alcohol treatment
– Not easily decolorize, stain holds “fast”)
Acid-Fast Stain
• Ziehl-Neelsen stain
– Carbol fuchsin - heat to penetrate
– Acid-alcohol - decolorize
– Methylene blue - counterstain
• Kinyon stain
– Carbol fuchsin – no heat, higher
phenol allow penetration
– Acid-alcohol - decolorize
– Methylene blue - counterstain
• Fluorochrome stain
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–
–
–
Auramine-rhodamine – stain, phenol
Acid-alcohol - decolorize
Acridine orange – counterstain
UV microscope scan slide high dry,
detect AFB by fluorescence
– Read stained slide easier, faster
Mycobacterium: Lab Culture
• Work under biosafety cabinet
• Specimen of choice
– Patient coughs up sputum from lung
– NaOH digest, decontaminate organic debris,
RT normal flora
– N-acetyl-L-cystine to liquify
• Enrich, selective media
– Egg or agar based
– Antimicrobial agents - malachite green,
cyclohexamide, nalidixic acid
• Lowenstein-Jensen medium – egg based,
colonies 18-24 days
• Middlebrook 7H10, 7H11 medium – agar
based, colonies 12-14 days
Mycobacterium tuberculosis:
Lab Culture
• Solid media - rough, dry, granular,
non-pigmented, buff color colony
• Liquid media - contains Tween 80,
albumin, faster growth
• RT grow best 5-10% CO2 , 370 C
• Skin lesion grow best, 30-330 C
• RT culture 6-8 weeks before
discard as negative
• Skin lesion culture 12 weeks
• CDC desires more timely report,
possible with new DNA
amplification methods of ID
Mycobacterium: Lab ID
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•
•
•
Rate of growth
Culture temperature
Pigmentation and photoreactivity
Biochemical testing: niacin production,
nitrate reduction, catalase at 680 C,
tween hydrolysis, arylsulfatase
production, tellurite reduction, salt
tolerance, pyrazinamidase production
• Test of choice - DNA amplification;
routinely done in PH lab, rapidly ID
species
Mycobacterium tuberculosis
• Human only natural reservoir
• Worldwide - third of population infected
– ~2 billion people
– ~8 million new cases/year
– ~2-3 million deaths/year
• USA ~10 million infected
– Since 1985, dramatic increase number
cases/year
– Infections in homeless, drug and alcohol
abusers, prisoners, AIDS patients
– After 1992, now slowly decline due to
increase PH prevention programs
M. tuberculosis: Virulence
Factors
• Cord factor – cell wall glycolipid
– Serpentine growth (filaments, cords), grow in
close parallel arrangement
– Toxic to leukocytes, anti-chemotactic
– Role in development of granulomatous lesions
• Iron capturing ability – required for
survival inside phagocytes
• Sulfolipids - prevent phagosome-lysosome
fusion (important in intracellular survival)
• Tissue damage - no known bacterial toxin
or enzyme implicated; host immune
response thought responsible, by
inflammation, cell-mediate immunity
(CMI)
M. tuberculosis: Transmission
• Close contact - person-to-person
• Inhalation - infectious aerosols into
alveolar spaces
• Exposure to few organisms (10-200)
may establish “infection” (elicit
immune response, no disease)
• Humans very susceptible to
infection, but remarkably resistant
to tuberculosis disease
Primary Tuberculosis
• Exposure - bacilli reach alveoli, ingested
by macrophage
• MO multiply - cause chemotactic
response, recruits macrophages, T cells
• Enzymes, cytokines release - start
inflammatory response, wall off MOs
(tubercle formation)
• Inflammatory response also causes lung
damage
• Small number MO - no tissue damage
• Large number MO - CMI response results
in tissue necrosis
• Patient becomes PPD skin test(+)
Early Tubercle
Reactivation Tuberculosis
• Few weeks macrophages die - release
bacilli, form caseous center in tubercle
• In healthy individuals - disease usually
arrested, lesions calcified
• Tubercle bacilli - may remain dormant in
lesion; later reactivation of disease
• Host defenses fail - mature tubercle
form; caseous center enlarge, liquify to
form tuberculous cavity where bacilli
multiply outside macrophage
Mature Tubercle
Extrapulonary Tuberculosis
• Tubercle ruptures - release bacilli;
disseminate throughout lung, circulatory,
lymphatic system
• Miliary (Extrapulmonary) TB – spread to
lymph nodes, pleura, many other organs:
– Progressive form of disease
– Weight loss, coughing with blood, loss of
vigor (old name consumption)
Tuberculosis: Infection and
Disease
• In 3-6 weeks - patient’s CMI activated,
bacteria replication stops
• Within 2 years - 5% patients progress to
active disease
• Sometime later in life - 5-10% patients
develop active disease
• AIDS patient, TB infected:
– Due to M. avian
– 10% develop active disease within 1 year
– 2x more likely to spread, rapidly progress to
death
– Impaired CMI unable to arrest infection
M. tuberculosis: Treatment
• Slow growth of MO, chronic infection
require 6-9 months drug treatment
• Combination of drugs to prevent
emergence of resistant strains
• Current recommended drugs: isoniazid
(INH), ethambutol, pyrazinamide,
rifampin
• Drug resistance
– 1990 report multidrug-resistant M. tb (MDRTB) in AIDS patients, homeless in N.Y., Miami
– In developing countries, extensively drugresistant TB (XDR-TB), resistance to second
line drugs, potentially untreatable
M. tuberculosis: Prevention
• BCG (bacille Calmette-Guerin)
Vaccine
– Attenuated M. bovis
– Used where TB high
– Reduce TB if vaccinated young age
• Tuberculin skin test
– PPD (purified protein derivative M.
tb) injected under skin
– Test host CMI response
– Delayed-type hypersensitivity
reaction (>10 mm induration), 48
hr., if previous or current infection;
not necessarily active disease
• Control disease
– PH surveillance
– Drug treatment and intervention
– Case monitoring, prevent
transmission
M. avium-intracellular
Complex
• Common in soil, water, food
• Before HIV - transient colonization in
patients with compromised pulmonary
function (bronchitis, obstructed
pulmonary disease, previous infection);
~pulmonary TB
• After HIV - USA most common
mycobacteria disease in AIDS patients
• Infection disseminated - all organs, large
number MO
M. avium-intracellular
Complex
• Transmission – ingestion of contaminated
food or water, not person-to-person
• Greatest risk for infection are
immunocompromised
• Multiply in localized lymph nodes, spreads
to disseminated disease
• Impair organ function due to replication
MOs, host immune response
• MO ubiquitous and control of exposure
difficult
Mycobacterium leprae:
Hansen’s Disease
• “to peel” “leprosy”
• ~12 M cases worldwide (Africa, Asia,
Latin America); rare USA
• Reservoir of MO in armadillo
• Does not grow in cell-free culture
• Transmission by person-to-person, direct
contact, inhale infectious aerosols
• Requires prolonged, intimate contact for
transmission
• Two clinical forms of disease:
– Tuberculoid
– Lepromatous
Hansen’s Disease
• Mycobacteria obligate intracellular
parasites in histiocytes, Schwann cells,
epitheloid structures - called Lepra cells
• Incubation period 2-4 years
• Clinical manifestations depend upon
adequacy of host CMI response
• Like TB - many infected, few develop
clinical symptoms of disease
• Subclinical/Tuberculoid – infection
contained by CMI
• Lepromatous - large number bacilli in
sputum, nasal secretion, skin
Hansen’s Disease: Tuberculoid
• Patient - strong CMI, weak
antibody response
• Lesions - skin, peripheral
nerves, few in number; raised,
erythematous margin, flat
center
• Nerve damage due to CMI
response - loss sensation of
touch, temperature; pain
within lesion
• Skin biopsy reveals many
lymphocytic, epithelial cells,
but no AFB
• Infectivity, transmission low
• Lepromin skin test (+)
Hansen’s Disease: Lepromatous
• Patient - strong antibody
response, defective CMI
(“foamy” macrophage, few
lymphocytes, numerous bacilli)
• Involve all areas of skin;
waxy, nodular appearance,
may thicken and fold
• Destruction of cutaneous
nerves, eyebrows, eyelashes,
nasal septum
• Skin biopsy reveals
lymphocytes, many Lepra cells
packed with AFB
• Infectivity high
• Lepromin skin test is (-)
M. leprae: Treatment and
Prevention
• Tuberculoid form – rifampicin,
dapsone; 6 months
• Lepromatous – rifampicin, dapsone,
clofazimine; minimum 1 year
• Control by prompt recognition and
treatment of infected patients
• Lepromin skin test is similar to TB
skin test
The Gifts of Civilization:
Germs and Genocide In Hawaii
• O. A. Bushnell, University of Hawaii Press.
1993
• Hawaii isolated, difficult to reach by sea
• 1778 – “discovered” by Captain Cook
• Estimate ~1 million Hawaiian inhabitants
• 1832 – census ~130,000
• 1900 - ~30,000
• Why decline?
• Infectious diseases upon immune naive
population i.e. STD, plague, cholera, TB,
Hansen’s disease from Chinese
immigrants, smallpox, chickenpox,
measles, mumps, rubella;
FATHER
DAMIEN
(1840 – 1889)
The man who lived and died
for the victims of Hansen’s
disease (leprosy)
Damien Born in Belgium
1840
• He was an
ordinary boy,
brother, and
priest.
• So what made him
different?
• Damien arrives
Hawaiian Islands
1864
• He agreed to do
a job that no one
else would do.
• He lived and
worked for a
group of people
who had been
sent away from
their homes to a
remote Hawaiian
island, Molokai
• They were outcasts because they had
Hansen’s Disease.
Hansen’s Disease
• A scaly skin
disease.
• A chronic disease
caused by a
bacteria.
• In those days there was no cure for
Hansen’s Disease.
• The outside world did not like to
think about this awful disease, and
chose to forget the people who
suffered from it.
• Damien gave back these people hope
and pride.
• He loved them like a family and in the
end he died as one of them, a victim
of Hansen’s Disease.
• Damien’s life and death forced people
to face the problem of Hansen’s
Disease.
• He said to the world that work
needed to be done – and quickly!
• Today, a cure for
Hansen’s Disease has
been found.
• We no longer have to
fear the disease or the
people who suffer from
it.
• In 2009, for Father
Damien’s many
contributions to
society, he was
Canonized Saint Damien
by the Vatican.
Class Assignment
• Textbook Reading
– Chapter 26 Mycobacterium Tuberculosis
– Omit: Clinical Significance and
Differentiation of Nontuberculosis
Mycobacterium
• Key Terms
• Learning Assessment Questions
Case Study 7: Mycobacterium
• A 35-year-old man with a history of
intravenous drug use entered the local
health clinic with complaints of a dry,
persistent cough; fever; malaise; and
anorexia.
• Over the preceding 4 weeks, he had lost
15 pounds and experienced chills and
sweats.
• A chest radiograph revealed patchy
infiltrates throughout the lung fields.
Case Study 7: Mycobacterium
• Because the patient had a nonproductive
cough, sputum was induced and submitted
for bacterial, fungal, and mycobacterial
cultures, as well as examination for
Pneumocystis organisms.
• Blood cultures and serologic tests for HIV
infection were performed.
• The patient was found to be HIV positive.
• The results of all cultures were negative
after 2 days of incubation; however,
cultures were positive for M. tuberculosis
after an additional week of incubation.
Case Study 7: Questions
• 1. What is unique about the cell wall of
mycobacteria, and what biologic effects
can be attributed to the cell wall
structure?
• 2. Why is M. tuberculosis more virulent in
patients with HIV infection than in nonHIV-infected patients?
• 3. What is the definition of a positive skin
test (PPD) result for M. tuberculosis?
• 4. Why do mycobacterial infections have
to be treated with multiple drugs for 6
months or more?