MYCOBACTERIUM

Mycobacteria are aerobic, acid-fast bacilli (rods).They are

neither gram-positive nor gram-negative (i.e., they are
stained poorly by the dyes used in Gram stain). They are
virtually the only bacteria that are acid-fast.
The term acid-fast refers to an organism’s ability to retain the
carbolfuchsin stain despite subsequent treatment with an
ethanol–hydrochloric acid mixture. The high lipid content
(approximately 60%) of their cell wall makes mycobacteria
acid-fast. The cell wall contains peptidoglycolipids, mycolic acids,
and other fatty acids and waxes; many of these compounds are
responsible for the various characteristics of mycobacteria (eg, slow
growth, acid fastness, resistance to detergents, and common
antibiotics).
The major pathogens are Mycobacterium tuberculosis, the cause of
tuberculosis, and Mycobacterium leprae, the cause of leprosy.
Atypical mycobacteria, such as Mycobacterium
avium-intracellulare complex and Mycobacterium kansasii,
can cause tuberculosis-like disease but are less frequent
pathogens.
Rapidly growing mycobacteria, such as Mycobacterium
chelonae, occasionally cause human disease in
immunocompromised patients or those in whom prosthetic
devices have been implanted.
 MYCOBACTERIUM TUBERCULOSIS
M. tuberculosis grows slowly (i.e., it has a doubling time of 18
hours, in contrast to most bacteria, which can double in number in 1
hour or less).
Because growth is so slow, cultures of clinical specimens must be
held for 6 to 8 weeks before being recorded as negative. M.
tuberculosis can be cultured on bacteriologic media, whereas M.
leprae cannot. Media used for its growth (e.g., Löwenstein-Jensen
medium) contain complex nutrients (e.g., egg yolk) and dyes (e.g.,
malachite green).The dyes inhibit the unwanted normal flora present
in sputum samples.
M. tuberculosis is an obligate aerobe; this explains
its predilection for causing disease in highly
oxygenated tissues such as the upper lobe of the lung
and the kidney.
The acid-fast property of M. tuberculosis (and other
mycobacteria) is attributed to long-chain (C78–C90)
fatty acids called mycolic acids in the cell wall.
Cord factor (trehalose
dimycolate) is correlated
with virulence of the
organism. Virulent strains
grow in a characteristic
“serpentine” cordlike
pattern, whereas avirulent
strains do not.
The organism also contains several proteins, which, when
combined with waxes, elicit delayed hypersensitivity. These
proteins are the antigens in the purified protein derivative
(PPD) skin test (also known as the tuberculin skin test).
A lipid located in the bacterial cell wall called phthiocerol
dimycocerosate is required for pathogenesis in the lung.
M. tuberculosis is relatively resistant to acids and alkalis.
NaOH is used to concentrate clinical specimens; it destroys
unwanted bacteria, human cells, and mucus but not the
organism.
M. tuberculosis is resistant to dehydration and therefore
survives in dried expectorated sputum; this property may be
important in its transmission by aerosol.
 Transmission & Epidemiology

M. tuberculosis is transmitted from person to person by

respiratory aerosol, and its initial site of infection is the
lung.
In the body, it resides chiefly within reticuloendothelial
cells (e.g., macrophages). Humans are the natural reservoir
of M. tuberculosis. Although some animals can be infected,
they are not a reservoir for human infection.
Most transmission occurs by aerosols generated by the coughing of
“smear-positive” people (i.e., those whose sputum contains
detectable bacilli in the acid-fast stain). However, about 20% of
people are infected by aerosols produced by the coughing of
“smear-negative” people.
The disease tuberculosis occurs in only a small number of infected
individuals. The risk of infection and disease is highest among
socioeconomically disadvantaged people, who have poor housing
and poor nutrition.*The estimated lifetime risk of developing
disease after primary infection is 10%, with roughly half of this
risk occurring in the first 2 years after infection.
Pathogenesis
 Pathogenesis of TB

M. tuberculosis produces
no exotoxins and does
not contain endotoxin in
its cell wall. In fact, no
mycobacteria produce
toxins.
 Laboratory Diagnosis

1- Acid-fast staining of sputum or other specimens is the

usual initial test (Figure 21– 1). Either the Kinyoun version
of the acid-fast stain or the older Ziehl-Neelsen version can
be used.
For rapid screening purposes, auramine stain, which can be
visualized by fluorescence microscopy, is used.
2- After digestion of the specimen by treatment with NaOH and
concentration by centrifugation, the material is cultured on special
media, such as Löwenstein-Jensen agar, for up to 8 weeks. It will not
grow on a blood agar plate. In liquid BACTEC medium, radioactive
metabolites are present, and growth can be detected by the production
of radioactive carbon dioxide in about 2 weeks. A liquid medium is
preferred for isolation because the organism grows more rapidly and
reliably than it does on agar. If growth in the culture occurs, the
organism can be identified by biochemical tests. For example, M.
tuberculosis produces niacin, whereas almost no other mycobacteria
do. It also produces catalase.
3- Nucleic acid amplification tests can be used to detect the
presence of M. tuberculosis directly in clinical specimens
such as sputum. Tests are available that detect either the
ribosomal RNA or the DNA of the organism. These tests
are highly specific, but their sensitivity varies. In sputum
specimens that are acid-fast stain positive, the sensitivity is
high, but in “smear-negative” sputums, the sensitivity is
significantly lower. These tests are quite useful in deciding
whether to initiate therapy prior to obtaining the culture
results.
Because drug resistance, especially to isoniazid , is a problem,
susceptibility tests should be performed. However, the organism
grows very slowly, and susceptibility tests usually take several
weeks, which is too long to guide the initial choice of drugs. To
address this problem, molecular tests are available, which detect
mutations in the chromosomal genes that encode either the catalase
gene that mediates resistance to isoniazid or the RNA polymerase
gene that mediates resistance to rifampin.
4-
4- The luciferase assay, which can detect drug-resistant
organisms in a few days, is also used. Luciferase is an
enzyme isolated from fireflies that produces flashes of light
in the presence of adenosine triphosphate (ATP). If the
organism isolated from the patient is resistant, it will not be
damaged by the drug (i.e., it will make a normal amount of
ATP), and the luciferase will produce the normal amount of
light. If the organism is sensitive to the drug, less ATP will
be made and less light produced.
 Diagnosis of Latent infection:

There are two approaches to the diagnosis of latent

infections.
1- One is the PPD skin test as. Because there are problems
both in the interpretation of the PPD test and with the
person returning for the skin test to be read, a quantifiable
laboratory-based test is valuable.
2- This laboratory test is an interferon-γ release assay (IGRA),
and there are two versions available: Quantiferon-TB and
T-spot.TB. In this assay, blood cells from the patient are exposed to
antigens from M. tuberculosis, and the amount of interferon-γ
released from the cells is measured. The sensitivity and specificity
of the IGRA are as good as the PPD skin test. Because the antigens
used in the test are specific for M. tuberculosis and are not present
in BCG, the test is not influenced by whether a person has been
previously immunized with the BCG vaccine or infected with
opportunistic mycobacteria .
Tuberculin Skin Test IGRA
BCG vaccine can be used to induce partial resistance to
tuberculosis. The vaccine contains a strain of live,
attenuated M. bovis called bacillus CalmetteGuérin.
The vaccine is effective in preventing the appearance of
tuberculosis as a clinical disease, especially in children,
although it does not prevent infection by M. tuberculosis.
However, a major problem with the vaccine is its variable
effectiveness, which can range from 0% to 70%. It is used primarily
in areas of the world where the incidence of the disease is high. It is
not usually used in the United States because of its variable
effectiveness and because the incidence of the disease is low
enough that it is not cost-effective.
The skin test reactivity induced by the vaccine given to children
wanes with time, and the interpretation of the skin test reaction in
adults is not altered by the vaccine.
 ATYPICAL MYCOBACTERIA

Several species of mycobacteria are characterized as

atypical, because they differ in certain respects from the
prototype, M. tuberculosis. For example, atypical
mycobacteria are widespread in the environment and are
not pathogenic for guinea pigs, whereas M. tuberculosis
is found only in humans and is highly pathogenic for
guinea pigs. The atypical mycobacteria are sometimes
called mycobacteria other than tuberculosis
 Group I (Photochromogens)

M. kansasii causes lung disease clinically resembling tuberculosis. Because it

is antigenically similar to M. tuberculosis, patients are frequently tuberculin
skin test– positive. Its habitat in the environment is unknown, but infections by
this organism are localized to the midwestern states and Texas. It is susceptible
to the standard antituberculosis drugs.
Mycobacterium marinum causes “swimming pool granuloma,” also known as
“fish tank granuloma.” These granulomatous, ulcerating lesions occur in the
skin at the site of abrasions incurred at swimming pools and aquariums. The
natural habitat of the organism is both fresh and saltwater. Treatment with a
tetracycline such as minocycline is effective.
 Group II (Scotochromogens)

M. scrofulaceum causes scrofula, a granulomatous

cervical adenitis, usually in children. (M.
tuberculosis also causes scrofula.) The organism
enters through the oropharynx and infects the
draining lymph nodes. Its natural habitat is
environmental water sources, but it has also been
isolated as a saprophyte from the human respiratory
tract. Scrofula can often be cured by surgical
excision of the affected lymph nodes.
 Group III (Nonchromogens)

M. avium-intracellulare complex (MAI, MAC) is composed of two species, M.

avium and M. intracellulare, that are very difficult to distinguish from each
other by standard laboratory tests. They cause pulmonary disease clinically
indistinguishable from tuberculosis, primarily in immunocompromised patients
such as those with AIDS who have CD4 cell counts of less than 200/mL. MAI
is the most common bacterial cause of disease in AIDS patients. The organisms
are widespread in the environment, including water and soil, particularly in the
southeastern United States. They are highly resistant to antituberculosis drugs,
and as many as six drugs in combination are frequently required for adequate
treatment. Current drugs of choice are clarithromycin plus one or more of the
following: ethambutol, rifabutin, or ciprofloxacin. Clarithromycin is currently
recommended for preventing disease in AIDS patients.
 Group IV (Rapidly Growing Mycobacteria)

Mycobacterium fortuitum-chelonae complex is composed of two

similar species, M. fortuitum and M. chelonae. They are
saprophytes, found chiefly in soil and water, and rarely cause human
disease. Infections occur chiefly in two populations: (1)
immunocompromised patients and (2) individuals with prosthetic
hip joints and indwelling catheters. Skin and soft tissue infections
occur at the site of puncture wounds (e.g., at tattoo sites). They are
often resistant to antituberculosis therapy, and therapy with multiple
drugs in combination plus surgical excision may be required for
effective treatment. Current drugs of choice are amikacin plus
doxycycline.
Mycobacterium abscessus is another rapidly growing
mycobacteria acquired from the environment. It causes chronic
lung infections, as well as infections of the skin, bone, and joints.
It is highly antibiotic-resistant. Mycobacterium smegmatis is a
rapidly growing mycobacterium that is not associated with
human disease. It is part of the normal flora of smegma, the
material that collects under the foreskin of the penis.
 MYCOBACTERIUM LEPRAE

This organism causes leprosy (Hansen’s disease).

 Important Properties
M. leprae has not been grown in the laboratory, either on artificial media or in cell
culture. It can be grown in experimental animals, such as mice and armadillos.
Humans are the natural hosts, although the armadillo appears to be a reservoir for
human infection in the Mississippi Delta region where these animals are common.
In view of this, leprosy can be thought of as a zoonotic disease, at least in certain
southern states, such as Louisiana and Texas.
The optimal temperature for growth (30°C) is lower than body temperature;
therefore, M. leprae grows preferentially in the skin and superficial nerves. It
grows very slowly, with a doubling time of 14 days. This makes it the
slowest-growing human bacterial pathogen. One consequence of this is that
antibiotic therapy must be continued for a long time, usually several years.
 Transmission
Infection is acquired by prolonged contact with patients with
lepromatous leprosy, who discharge M. leprae in large numbers in nasal
secretions and from skin lesions. In the United States, leprosy occurs
primarily in Texas, Louisiana, California, and Hawaii. Most cases are
found in immigrants from Mexico, the Philippines, Southeast Asia, and
India. The disease occurs worldwide, with most cases in the tropical
areas of Asia and Africa. The armadillo is unlikely to be an important
reservoir because it is not found in many areas of the world where
leprosy is endemic.
 Pathogenesis

The organism replicates intracellularly, typically within skin

histiocytes, endothelial cells, and the Schwann cells of nerves.
The nerve damage in leprosy is the result of two processes:
damage caused by direct contact with the bacterium and damage
caused by CMI attack on the nerves.
There are two distinct forms of leprosy—tuberculoid and
lepromatous—with several intermediate forms between the two
extremes.
In tuberculoid (also known as paucibacillary) leprosy, the CMI response
to the organism limits its growth, very few acid-fast bacilli are seen,
and granulomas containing giant cells form. The nerve damage seems
likely to be caused by cell mediated immunity as there are few
organisms and the CMI response is strong. The CMI response consists
primarily of CD4-positive cells and a Th-1 profile of cytokines, namely,
interferon-γ, interleukin-2, and interleukin-12. It is the CMI response
that causes the nerve damage seen in tuberculoid leprosy. The lepromin
skin test result is positive. The lepromin skin test is similar to the
tuberculin test. An extract of M. leprae is injected intradermally, and
induration is observed 48 hours later in those in whom a CMI response
against the organism exists.
In lepromatous (also known as multibacillary) leprosy, the cell-mediated response to
the organism is poor, the skin and mucous membrane lesions contain large numbers of
organisms, foamy histiocytes rather than granulomas are found, and the lepromin skin
test result is negative. The nerve damage seems likely to be caused by direct contact as
there are many organisms and the CMI response is poor. There is evidence that people
with lepromatous leprosy produce interferon-β (antiviral interferon) in response to M.
leprae infection, whereas people with tuberculoid leprosy produce interferon-γ.
Interferon-β inhibits the synthesis of interferon-γ thereby reducing the CMI response
needed to contain the infection. Note that in lepromatous leprosy, only the
cell-mediated response to M. leprae is defective (i.e., the patient is anergic to M.
leprae). The cell-mediated response to other organisms is unaffected, and the humoral
response to M. leprae is intact. However, these antibodies are not protective. The T-cell
response consists primarily of Th-2 cells.
 Clinical Findings

Tuberculoid leprosy. The tuberculoid form is characterized by a single, flat,

hypopigmented lesion that has lost sensation.
Lepromatous leprosy. The lepromatous form is characterized by multiple,
raised lesions, often with the appearance of leonine facies.
 Laboratory Diagnosis

In lepromatous leprosy, the bacilli are easily demonstrated by performing

an acid fast stain of skin lesions or nasal scrapings. Lipid-laden
macrophages called “foam cells” containing many acid-fast bacilli are seen
in the skin.
In the tuberculoid form, very few organisms are seen, and the appearance
of typical granulomas is sufficient for diagnosis.
Cultures are negative because the organism does not grow on artificial
media.
A serologic test for IgM against phenolic glycolipid-1 is useful in the
diagnosis of lepromatous leprosy but is not useful in the diagnosis of
tuberculoid leprosy. The diagnosis of lepromatous leprosy can be
confirmed by using the polymerase chain reaction (PCR) test on a
skin sample.
False-positive results in the nonspecific serologic tests for syphilis,
such as the Venereal Disease Research Laboratory (VDRL) and rapid
plasma reagin (RPR) tests, occur frequently in patients with
lepromatous leprosy.
 Treatment
The mainstay of therapy is dapsone (diaminodiphenylsulfone), but because sufficient
resistance to the drug has emerged, combination therapy is now recommended (e.g.,
dapsone, rifampin, and clofazimine for lepromatous leprosy and dapsone and
rifampin for the tuberculoid form). Treatment is given for at least 2 years or until the
lesions are free of organisms. A combination of ofloxacin plus clarithromycin is an
alternative regimen. Thalidomide is the treatment of choice for severe ENL reactions.
 Prevention

Isolation of all lepromatous patients, coupled with

chemoprophylaxis with dapsone for exposed children, is
required.
There is no vaccine.