Mycobacteriaceae
Mycobacteriaceae
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Classification –
Family Mycobacteriaceae
 1 genus of medical importance=
Mycobacteria
 All are slow growing
 All are acid-fast and contain large amounts
of lipids in their cell walls
 Tubercle bacilli= M. tuberculosis, M.
africanum, and M. bovis
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Mycobacteriaceae
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Mycobacteria other than tubercle bacilli (MOTT) or the
atypicals= all other Mycobacteria species
The Mycobacteria are sometimes divided into 4 groups
(Runyon groups) based on growth rate and pigmentation:
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Photochromagens – are non-pigmented when grown in the
dark, but produce photoactivated pigments upon exposure
to light
Scotochromogens – produce deep yellow to orange
pigments when grown in light or dark, but the color will
deepen upon two weeks exposure to light
Nonphotochromagens – may produce pigment ranging from
white to yellow, but pigment does not intensify upon
exposure to light. M. tuberculosis belongs in this group.
Rapid growers – organisms that form colonies within seven
days.
Mycobacteriaceae
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Mycobacterium leprae does not fall into any of
these groups and it can’t be grown on ordinary lab
media.
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It is grown in the 9 banded armadillo or in the footpads of
mice (takes 3-4 weeks to replicate!)
Morphology and cultural characteristics
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Always work under a biosafety cabinet!
Slender G+ rods that stain poorly because of the
large amount of lipids in the cell wall.
Are all acid-fast – the acid-fast stain is an important
first step for presumptive diagnosis of
mycobacterial disease.
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A concentrated specimen is often used.
Mycobacteriaceae
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Heating is required for stain penetration because
of the high lipid content of the cell wall (mycolic
acid and waxD).
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Once the stain penetrates and binds to the mycolic
acid in the cell wall, it cannot be removed even with
acid alcohol treatment.
Mycobacteriaceae
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There are several different acid fast stains that
may be used:
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Ziehl-Neelsen – uses heat to get the primary stain of
carbol fuchsin to penetrate the cell wall; acid alcohol
destaining; and methylene blue as the counterstain.
Kinyon – uses a higher content of phenol (organic
solvent) in the carbol fuchsin primary stain to allow
penetration of the stain without the need to apply heat.
 Also uses acid alcohol to destain and methylene
blue as the counterstain.
Acid-fast bacilli
Mycobacteriaceae
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Auramine-rhodamine fluorochrome (a fluorescent
stain) requires a fluorescent microscope, but allows
one to scan the slide a high dry so that slides may be
read much faster
 Stain with auramine-rhodamine for 10 minutes
(phenol in the solution allows for penetration)
 Destain with acid alcohol
 Counterstain with acridine orange
 A positive result is a bright yellow fluorescence.
Reporting results: 1-2 AFB/300 fields +/-; 1-9
AFB/100 fields 1+
Mycobacteriaceae
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If the specimen is highly contaminated with organic
debris and normal flora, before plating the
specimen should be digested and decontaminated
with NaOH
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Addition of N-acetyl-L-cystine helps to liquify sputum.
Most will grow on simple media, but for primary
isolation complex media should also be used – use
of a nonselective, a selective and possibly a liquid
media is recommended.
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Nonselective – may be egg or agar based and may
include malachite green to suppress growth of
contaminating bacteria.
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Lowenstein-Jensen – egg based; colonies grow in 18-24
days)
Mycobacteriaceae
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Middlebrook 7H10 and 7H11 – agar based; colonies
grow in 12-14 days.
Selective media – consists of one of the
nonselective media plus added antimicrobial
agents (malachite green, cyclohexamide, and
nalidixic acid are often used)
 The colonies of M. tuberculosis on the solid
media are rough, dry, granular, nonpigmented to
buff or tan colored colonies.
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M. tuberculosis culture
Mycobacteriaceae
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Liquid media - the media usually contains tween 80 and albumin
and the organisms will grow faster than on solid media
Most Mycobacteria grow best in 5-10% CO2 and at 35-370 C.
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Cultures should be kept 6-8 weeks before being discarded as 
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Organisms from skin lesions grow best at 30-330 C.
Skin lesion cultures should be kept for 12 weeks.
CDC wants results in 1-2 weeks and this is only possible with
the new PCR based methods of identification.
Identification
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Rate of growth and growth in relation to temperature
Pigmentation and photoreactivity
Further biochemical testing includes:
Mycobacteriaceae
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Niacin – a niacin + slow growing organism producing
rough, dry, granular colonies that resemble Tb is strongly
presumptive of Tb, but it must be differentiated from M.
simiae
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M. tb. Is nitrate reduction+ and – for catalase at 680 C
M. simiae is – for nitrate reduction and + for catalase at 680
C.
M simiae is also a photochromagen, but its photoreactivity
may be delayed.
Tween hydrolysis, arylsulfatase production, tellurite
reduction, salt tolerance, and pyrazinamidase production
may also be used in differentiating the different species of
Mycobacteria.
Mycobacteriaceae
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Virulence factors (M. tb.)
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Cord factor – is a glycolipid, trehalose 6,6’
dimycolate that is responsible for the serpentine
growth (filaments or cords) of M. tb. in which the
bacilli grow in close parallel arrangement.
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It is toxic to leukocytes, activates macrophages and
dendritic cells, interferes with mitochondrial function in
mice and plays a role in the development of
granulomatous lesions
Iron capturing ability – required for survival inside
phagocytes
Sulfolipids prevent phagosome-lysosome fusion so
that the organisms are not exposed to lysosomal
enzymes (important in intracellular survival)
Mycobacteriaceae
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Clinical significance (M.tb.)
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Both M. tuberculosis and M. bovis can cause Tb,
but M. bovis is rare in the U.S.
The organism can gain entrance by inhalation
(most common), ingestion (usually from
contaminated milk), or through mucous membranes
of the genital-urinary tract or conjunctiva, or through
skin abrasions.
Man is very susceptible to infection, but remarkably
resistant to tuberculous disease.
Mycobacteriaceae
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Progression of the infection after inhalation:
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Tubercle bacilli that reach the alveoli of the lung are
ingested by macrophages
The organisms multiply and cause a chemotactic
response that recruits other macrophages and T cells to
the area.
Enzymes and cytokines are released to start an
inflammatory response to wall off the organism (tubercle
formation), but the inflammatory response also causes
lung damage.
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At his point the patient becomes skin test +.
Mycobacteriaceae
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After a few weeks many of the macrophages die, releasing
tubercle bacilli and forming a caseous center in the
tubercle.
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In healthy individuals, the disease is usually arrested at this
time and the lesions may may become calcified (Ghon
complexes).
Tubercle bacilli may remain dormant in the lesion and serve
as a basis for later reactivation of the disease.
When the defenses fail, a mature tubercle may form
whereby the caseous center will enlarge and liquify to form
a tuberculous cavity where the bacilli multiply outside the
macrophages.
Mycobacteriaceae
The tubercle eventually ruptures, releasing
tubercle bacilli that can disseminate throughout
the lungs and then to the circulatory and
lymphatic systems (Miliary Tb).
 This is the progressive form of the disease and it
is characterized by weight loss, coughing with
blood, and loss of vigor (The old name for this
disease was consumption because it is as if the
body is being consumed by the bacteria)
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Progression of tuberculosis
Progression of tuberculosis
Progression of tuberculosis
Mycobacteriaceae
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Clinical significance – the atypicals
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Are usually transmitted form soil or water to man
Some produce pulmonary diseases that resemble
Tb and others cause skin lesions
The M. avium-intracellulare group has become a
major problem in AIDs patients where the
organisms may be cultured from the blood and
stools.
Clinical significance – M. leprae
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Causes leprosy which is also called Hansen’s
disease
Mycobacteriaceae
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The mycobacteria are obligate intracellular
parasites found in histiocytes, Schwann cells, and
in epitheloid structures called Lepra cells
The mode of transmission is unknown, but in
lepromatous leprosy there are large numbers of
bacilli in the sputum and nasal secretions
The disease is contagious, but requires prolonged,
intimate contact for transmission
The incubation is 2-4 years
The clinical manifestations depend upon the
adequacy of the host cell mediated response
against the organism.
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Like Tb, many are infected, but few develop clinical
symptoms.
Mycobacteriaceae
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Subclinical – the infection is contained by the cell
mediated immune response
Tuberculoid – this form of the disease occurs when the cell
mediated immune response is partially effective:
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Lesions are confined to the skin and peripheral nerves
Lesions are solitary or few in number
Lesions have a raised, erythematous margin and a flat
center
Nerve damage due to the cell mediated immune response
causes a loss of sensation to touch, temperature, and pain
within the lesion
A skin biopsy reveals many lymphocytic and epithelial cells,
but no AFB.
The lepromin test is +
Tuberculous form of leprosy
Mycobacteriaceae
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Lepromatous – this form of the disease occurs
when there is defective cell mediated immunity
against the organism
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May involve all areas of the skin and lesions have a
waxy, nodular appearance
There is destruction of cutaneous nerves
The skin may thicken and fold
There is a loss of eyebrows and eyelashes
There may be destruction of the nasal septum
A skin biopsy reveals no lymphocytes and many Lepra
cells packed with AFB.
The lepromin test is -
Lepromatous form of leprosy
Lepromatous form of leprosy
Lepromatous form of leprosy
Lepromatous form of leprosy
Mycobacteriaceae
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Indeterminate
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There is a hypopigmented macule
There may or may not be sensory loss within the
macule
The macule may completely resolve
Reactionary state
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Erythema nodosum leprosum (ENL)
 This is an antibody mediated reaction in which
immune complexes form
 Painful, erythematous nodules form
 May occur in individuals with the lepromatous
form of the disease following drug therapy
Mycobacteriaceae
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Lucio phenomena – necrotizing skin lesions which blister
and ulcerate may occur.
Treatment -Tb
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Use a combination of drugs to help prevent the
emergence of resistant strains
Isoniazid
Rifampin
Para-aminosalicylic acid
Ethambutol
Streptomycin
Antibiotics must be given over an extended period
of time – at least 9 months
Mycobacteriaceae
BCG is a preventative vaccine (not used in
the U.S.). It was made by passaging the
organism in culture over a long period of
time to attenuate it.
 Tuberculin skin test – the Mantoux test uses
PPD (purified protein derivative) which is
injected just under the skin.
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A delayed-type hypersensitivity reaction
indicates a previous or current infection, but
does not mean that you have the disease.
 If results are +, X-ray for Ghon complexes
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Tuberculin skin test
Ghon complexes
Mycobacteriaceae
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Treatment – atypicals
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Must use multiple drugs because resistance is common
Teatment – leprosy
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Dapsone –
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For those with the lepromatous form of the disease, treatment is
for life
For those with the tuberculoid form of the disease, treatment is
for 5-10 years (Note that treatment may cause the development
of ENL)
Rifampin
Clofazimine
Thalodimide – treats ENL symptoms
Lepromin skin test is similar to the Tb skin test