MYCOBACTERIA
Mycobacterium are aerobic, non–spore-
forming (except for Mycobacterium
marinum and M. avium subsp.
paratuberculosis), nonmotile, very thin,
slightly curved or straight rods
Mycobacterium is the only genus in the
Mycobacteriaceae
Genera that are closely related to
Mycobacterium include Corynebacterium,
Nocardia, Rhodococcus, Segniliparus,
Tsukamurella, and Gordonia
Mycobacterium spp. have an unusual cell
wall; it contains N-glycolylmuramic acid
instead of N-acetylmuramic acid and has a
very high lipid content, which creates a
hydrophobic permeability barrier.
Mycobacteria are difficult to stain with Gram
staining, but are generally considered gram
positive.
acid fastness; this characteristic
distinguishes mycobacteria from other
genera.
Rapid-growing mycobacteria (RGM), in
which growth is apparent sooner than 7
days after subculture to LowensteinJensen
medium, may partially or completely lose
this characteristic as a result of their growth
characteristics.
Another important feature of many species
is that they grow more slowly than most
other human pathogenic bacteria because
of their hydrophobic cell surface.
Slow-growing mycobacteria, by definition,
require more than 7 days to produce
colonies on solid media.
The genus Mycobacterium includes several
highly pathogenic species included in the M.
tuberculosis complex—M. uclerans and the
nonculturable M. leprae. There are also
more than 180 species of nonculturable
nontuberculosis mycobacteria (NTM), many
of which are increasingly found in
opportunistic pathogens that are found
throughout the environment.
For the most part, mycobacteria can be
divided into two major groups based on
fundamental differences in epidemiology
and association with disease: those
belonging to the M. tuberculosis complex
and the others to the NTM group.
I. MYCOBACTERIUM TUBERCULOSIS
COMPLEX (MTC)
TUBERCULOSIS / COMSUMPTION
EPIDEMIOLOGY AND PATHOGENESIS
EPIDEMIOLOGY
 M. tuberculosis is the cause of most cases
of human tuberculosis, particularly in
developed countries.
 M. tuberculosis complex organisms are not
able to replicate in the environment and are
therefore isolated to growth in tissues of
humans and other warm-blooded animals.
Tuberculosis continues to be a public health
problem in the United States. An additional
complicating factor in the management of
tuberculosis is the increasing incidence of
coinfection with HIV. HIV-associated
tuberculosis
In the United States, tuberculosis is typically
found among the poor, the homeless,
intravenous drug users, alcoholics, the
elderly—in general, medically underserved
populations.
Another area of concern is the increased
identification of multidrug-resistant
tuberculosis (MDR-TB) and extensively
drug-resistant tuberculosis (XDR-TB).
Pathogenesis
Inhalation of a single viable organism has
been shown to lead to infection, although
close contact is usually necessary.
Of those who become infected with M.
tuberculosis, 15% to 20% develop disease.
The disease usually occurs some years
after the initial infection, when the patient’s
immune system breaks down for some
reason other than the presence of
tuberculosis bacilli in the lung. In a small
percentage of infected hosts, the disease
 becomes systemic, affecting a variety of
organs.
INGESTION OF UNPASTEURIZED MILK
cows, M. bovis inhalation of infectious
droplets from infected cattle.
An attenuated strain of M. bovis, bacillus
Calmette-Guérin (BCG), has been used
extensively in many parts of the world to
immunize susceptible individuals against
tuberculosis.
M. africanum demonstrates physiologic and
biochemical properties that place it
intermediately between M. tuberculosis and
M. bovis. This organism has been primarily
identified as the cause of approximately half
of the cases of tuberculosis in West Africa,
but it has also been found in the United
States in patients who previously resided in
Africa.
M. caprae can be identified by its
susceptibility to pyrazinamide. This
organism is associated with approximately
31% of the cases of human tuberculosis.
Reservoir hosts for the organism include
goats, cattle, sheep, pigs, wild boars, deer,
and fox.
M. microti, typically found in rodents, guinea
pigs, rabbits, cats, llamas, and meerkats,
usually fails to grow in culture.
It has been identified in tuberculosis in both
immunocompetent and immunosuppressed
patients. M. canettii has been primarily
identified in cases of lymphadenitis and
generalized tuberculosis in
immunocompromised individuals.
M. pinnipedii is transmitted from sea lions to
humans and has been associated with
granulomatous lesions in the lymph nodes,
lungs, pleura, and spleen. It is also
pathogenic in guinea pigs, rabbits,
camels, and possibly cattle.
The two additional species in the M.
tuberculosis complex reservoir hosts include
the banded mongoose (M. mungi) or large
mammals such as gazelles, antelopes,
waterbucks, and oryxes (M. orygis).
SPECTRUM OF DISEASE
 Tuberculosis may mimic other diseases,
such as pneumonia, neoplasm, or fungal
infections.
 In addition, clinical manifestations in
patients infected with M. tuberculosis
complex may range from asymptomatic to
acutely symptomatic.
 Cases of pulmonary disease caused by M.
tuberculosis complex organisms are
clinically, radiologically, and pathologically
indistinguishable
 Primary tuberculosis typically is
considered a disease of the respiratory
tract. Common presenting symptoms
include low-grade fever, night sweats,
fatigue, anorexia (loss of appetite), and
weight loss.
 These organs include the following:
o Genitourinary tract
o Lymph nodes (cervical lymphadenitis)
o Central nervous system (meningitis)
o Bone and joint (arthritis and osteomyelitis)
o Peritoneum
o Pericardium
o Larynx
o Pleural lining (pleuritis)
 Disseminated tuberculosis can be
diagnosed by a positive tuberculin skin
test
 Patients may also have latent tuberculosis
 A patient with latent tuberculosis is not
infectious and does not have active disease,
although the organism is present in
granulomas. Patients with latent
tuberculosis may progress to active disease
( reactivation tuberculosis) at any time.
 Reactivation tuberculosis typically occurs
after an incident in which cellular immunity
is suppressed or damaged as a result of a
change in lifestyle or other health condition.
Individuals infected with HIV are particularly
susceptible to developing active
tuberculosis.
Diagnosing tuberculosis is more difficult in
people infected with HIV because are
anergic (lack a biologic response) to
tuberculin skin testing, a primary means
of identifying individuals infected with M.
tuberculosis.
 The tuberculin skin test, or purified
protein derivative (PPD) test, is based on
the premise that after infection with M.
tuberculosis, an individual develops a
delayed hypersensitivity cell-mediated
immunity to certain antigenic components of
the organism.
After 48 to 72 hours, an infected individual
will show a delayed hypersensitivity reaction
to the PPD, characterized by erythema
(redness) and, most important, induration
(firmness because of the influx of immune
cells
The PPD test is not 100% sensitive or
specific, and a positive reaction to the skin
test does not necessarily signify the
presence of disease.

The T-Spot TB test

enzyme-linked immunosorbent assay
(ELISA) called QuantiFERON-TB Gold Plus
2. NONTUBERCULOUS MYCOBACTERIA
a. Slow-Growing
b. Rapidly Growing NTM and;
c. Non-cultivatable NTM
The NTM include all mycobacterial species
that do not belong to the M. tuberculosis
complex. Currently approximately 180
species
 The members of this large group of
mycobacteria are often opportunistic
pathogens.
 Significant geographic variability is seen
both in the prevalence of and the species
responsible for NTM disease.
 present everywhere in the environment and
sometimes colonize the skin and respiratory RUNYON GROUPS I TO IV
and gastrointestinal tracts of healthy
RUNYON GROUPS I SLOW-GROWING
individuals TO III NTM
 Interpretation of a positive NTM culture is RUNYON GROUP IV rapid growers NTM
complicated.
  Mycobacterium spp. synthesize
carotenoids (a group of yellow to red
pigments) in varying amounts and thus can
be categorized into three groups—
photochromogens, scotochromogens,
and nonphotochromogens—based on the
production of those pigments
The SLOW-GROWING NTM CAN BE
SUBDIVIDED INTO THREE GROUPS based
on the production of those pigments

A. PHOTOCHROMOGENS
The photochromogens (Table 42.3) are slow-
growing NTM that produce colonies requiring
light to form pigment.
B. SCOTOCHROMOGENS
The scotochromogens (Table 42.4) are slow-
growing NTM that produce pigmented
colonies whether grown in the dark or the
light
B. NONPHOTOCHROMOGENS
The nonphotochromogens (Table 42.5) are
slow-growing NTM that produce unpigmented
colonies whether grown in the dark or the
A. SLOW-GROWING NONTUBERCULOUS
light
MYCOBACTERIA
MYCOBACTERIUM AVIUM COMPLEX
The introduction of highly active
antiretroviral therapy (HAART) has
reduced the infections caused by M. avium
complex in patients with AIDS.
GENERAL CHARACTERISTICS.
 MAC comprises M. avium, Mycobacterium
intracellulare, M. avium subsp. avium, M.
avium subsp. paratuberculosis, M. avium
subsp. silvaticum (wood pigeon bacillus), M.
avium subsp. hominissuis, M. arosiense, M.
vulneris, M. marseillense, M.
bouchedurhonense, M. chimaera, M.
colombiense, M. yogonense, and M.
timonense.
EPIDEMIOLOGY AND PATHOGENESIS
 MAC is an important pathogen in both
immunocompromised and
immunocompetent populations.
 MAC is particularly noteworthy for its
potentially pathogenic role in pulmonary
infections in patients with AIDS and in those
who are not infected with HIV. T
 organisms are ubiquitous in the
environment and have been isolated from
natural water, soil, dairy products, pigs,
chickens, cats, and dogs.
 Infections caused by MAC are acquired by
inhalation or ingestion.
 NTM have extraordinary starvation survival.  MAC cultures can have an opaque glossy-
white colony morphology or can produce a
smaller translucent colony morphology
CLINICAL SPECTRUM OF DISEASE.

B. RAPIDLY GROWING potentially pathogenic species based on

NONTUBERCULOUS MYCOBACTERIA pigmentation and molecular studies.
 The common human pathogens in this
 The large group of organisms that constitute
group include Mycobacterium abscessus
the RGM is divided into six major groups of
 subsp. abscessus, Mycobacterium
chelonae, and Mycobacterium fortuitum.
On Gram staining, these organisms appear
as weakly gram-positive rods resembling
diphtheroids.
EPIDEMIOLOGY AND PATHOGENESIS
 The rapidly growing mycobacteria
considered potentially pathogenic can
cause disease in either healthy or
immunocompromised patients.
 Like many other NTM, these organisms are
ubiquitous in the environment and are
present worldwide.
 They have been found in soil, marshes,
rivers, and municipal water supplies (tap
water) and in marine and terrestrial life
forms.
 may be commensals on the skin.
 They can also be health care–associated
infections
SPECTRUM OF DISEASE
 The most common infection associated with
RGM is posttraumatic wound infection.
 C. NONCULTIVATABLE
NONTUBERCULOUS MYCOBACTERIA—
MYCOBACTERIUM LEPRAE
 The nontuberculous mycobacterium M.
leprae is a close relative of M. tuberculosis.
 M. leprae has not yet been cultivated
This organism causes leprosy (also called
Hansen disease)
 Hansen disease/ Leprosy is a chronic
disease of the skin, mucous membranes,
and nerve tissue. Leprosy remains a
worldwide public health concern owing to
the development of drug-resistant isolates.
in vitro, although it can be cultivated in the
armadillo and in the footpads of mice.
 Molecular biologic techniques have
provided most of the information about this
organism’s genomic structure and its
various genes and their products.
 Routine diagnosis of leprosy is based on
distinct clinical manifestations, such as
hypopigmented skin lesions and peripheral
nerve involvement, in conjunction with a
skin smear that tests positive for acid-fast
bacilli (AFB).
EPIDEMIOLOGY AND PATHOGENESIS
Understanding of the epidemiology and
pathogenesis of leprosy is hampered by the
inability to grow the organism in culture.
EPIDEMIOLOGY
 The primary reservoir for M. leprae is
infected humans.
 The disease is transmitted person to person
through inhalation or contact with infected
skin.
PATHOGENESIS
 leprosy is both a bacterial and an
immunologic disease.
Two primary phases are a silent phase,
during which the leprosy bacilli multiply in
the skin in macrophages, and an
intermediate phase, in which the bacilli
multiply in peripheral nerves and begin to
cause sensory impairment.
SPECTRUM OF DISEASE
 spectrum of disease caused by M. leprae
ranges from subclinical infection to
intermediate stages of disease to full-blown
and serious clinical manifestations involving
the skin, upper respiratory system, testes,
and peripheral nerves. T
 Two major forms of the disease are a
localized form, called 1. tuberculoid
leprosy, and a more disseminated form,
called 2. lepromatous leprosy.
Patients with lepromatous leprosy are
anergic to M. leprae because of a defect in
their cell-mediated immunity. Because the
organisms’ growth is unimpeded, these
individuals develop extensive skin lesions
containing numerous AFB; the organisms
can spill over into the blood and
disseminate.
In contrast, individuals with tuberculoid
leprosy do not have an immune defect; the
disease is localized to the skin and nerves.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTION
Purpose of AFB Test
 TB diagnosis
 Other mycobacterial infection detection
 Monitoring treatment response
 Identifying the cause of symptoms
SPECIMEN COLLECTION AND TRANSPORT
1. Pulmonary spx
2. Gastric Lavage spx
3. Urine Spx
4. Fecal spx
5. Tissue and Body Fluid
6. Blood spx
7. Wounds, Skins lesions, and Aspirates
SPECIMEN PROCESSING
CONTAMINATED SPECIMENS
 Most specimens submitted for
mycobacterial culture consist of organic
debris, such as mucin, tissue, serum, and
other proteinaceous material contaminated
with organisms.
 Rapidly growing mycobacteris are
especially susceptible to high or prolonged
exposure to 2% or more sodium hydroxide
(NaOH)
 Digestion-decontamination procedures
should be as gentle as possible.
INADEQUATE SPECIMEN AND REJECTION
CRITERIA
 Specimens should be rejected according to
the following guidelines: (1) insufficient
 volume. (2) contamination with saliva, (3)  NaOH method
dried swabs, (4) pooled sputum or urine, (5)  Zephiran-trisodium phosphate method
container has been compromised or is  N-acetyl-L-cysteine (NALC)-2% NaOH
broken or leaking, and (6) length of time method
from collection to processing is too long  Oxalic acid

SPECIAL CONSIDERATIONS  For fecal specimens, approximately 0.2 g of

stool is emulsified in 11 mL of sterile,
 Aerosol-induced sputum should be treated
filtered, distilled water.
as sputum
 Swabs and wound aspirates should be
 Gastric lavages should be processed within
transferred to a sterile, 50-ml. conical
4 hours of collection or neutralized with 10%
centrifuge tube containing a liquid medium
sodium carbonate and refrigerated until
at a ratio of 1 part specimen to 5 to 10 parts
processed as for sputum.
liquid medium.
 Urine specimens should be divided into a
 Large pieces of tissue should be finely
maximum of four 50-mL centrifuge tubes
minced with a sterile scalpel and scissors.
and centrifuged at 36003 g for 30 minutes.
 1. SOLID MEDIA

SPECIMENS NOT REQUIRING DECONTAMINATION

 Tissues or body fluids collected aseptically usually

do not require the digestion and decontamination
methods used with contaminated specimens.
 + CSF should be handled aseptically and
centrifuged for 30 minutes at 36003 g to
concentrate the bacteria.
 Pleural fluid should be collected in sterile
anticoagulant (1 mg/mL
ethylenediaminetetraacetic acid [EDTA] or 0.1
mg/mL, heparin).
 Joint fluid and other sterile exudates can be
handled aseptically and inoculated directly to
media.
 Bone marrow aspirates may be injected into
Pediatric Isolator tubes (Alere, Waltham, MA),
which help prevent clotting

DIRECT DETECTION
1. Microscopy
2. Acid Fast Stain
3. Fluorochrome and Fuschin and Acid Fast
4. Antigen-Protein Detection
5. Immunodiagnostic Testing
6. Genetic Sequence and Nuclear Acid
Amplification
2. LIQUID MEDIA

CULTIVATION
APPROACH TO IDENTIFICATION