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Official reprint from UpToDate

www.uptodate.com 2015 UpToDate

Natural history, microbiology, and pathogenesis of tuberculosis

Author
Lee W Riley, MD

Section Editor
C Fordham von Reyn, MD

Deputy Editor
Elinor L Baron, MD, DTMH

All topics are updated as new evidence becomes available and our peer review process is complete.
Literature review current through: Jul 2015. | This topic last updated: Mar 10, 2015.
INTRODUCTION Mycobacterium tuberculosis is the second most common infectious cause of death in adults
worldwide (HIV is the most common). The human host serves as a natural reservoir for M. tuberculosis. The ability
of the organism to efficiently establish latent infection has enabled it to spread to nearly one-third of individuals
worldwide. Approximately 8 million new cases of active TB disease occur each year, leading to about 1.7 million
deaths. The disease incidence is magnified by the concurrent epidemic of human immunodeficiency virus (HIV)
infection. (See "Epidemiology of tuberculosis".)
The microbiology and pathogenesis of M. tuberculosis will be reviewed here. The immunology of this infection is
discussed separately. (See "Immunology of tuberculosis".)
NATURAL HISTORY OF INFECTION Inhalation of aerosol droplets containing M. tuberculosis with subsequent
deposition in the lungs leads to one of four possible outcomes:
Immediate clearance of the organism
Primary disease: immediate onset of active disease
Latent infection
Reactivation disease: onset of active disease many years following a period of latent infection
Among individuals with latent infection and no underlying medical problems, reactivation disease occurs in
approximately 5 to 10 percent of cases [1-3]. The risk of reactivation is markedly increased in patients with HIV and
other medical conditions [4,5]. These outcomes are determined by the interplay of factors attributable to both the
organism and the host.
Primary disease Much of our understanding of the natural course of tuberculosis (TB) comes from human
autopsy data prior to the era of antituberculosis drugs and from experimental animal models [6-10]. Among the
approximately 5 to 10 percent of infected individuals who develop active disease, approximately half will do so within
the first two to three years following infection.
The tubercle bacilli establish infection in the lungs after they are carried in droplets small enough to reach the
alveolar space (5 to 10 microns). If the innate defense system of the host fails to eliminate the infection, the bacilli
proliferate inside alveolar macrophages, which may migrate away from the lungs to enter other tissue.
While in the lungs, macrophages produce cytokines and chemokines that attract other phagocytic cells, including
monocytes, other alveolar macrophages, and neutrophils, which eventually form a nodular granulomatous structure
called a tubercle. If the bacterial replication is not controlled, the tubercle enlarges and the bacilli enter local draining
lymph nodes. This leads to lymphadenopathy, a characteristic manifestation of primary TB. The lesion produced by
the expansion of the tubercle into the lung parenchyma and lymph node involvement is called the Ghon complex.
Bacteremia also may accompany initial infection.
The bacilli continue to proliferate until an effective cell-mediated immune (CMI) response develops, usually 2 to 10
weeks following initial infection; this occurs in more than 90 percent of exposed infected individuals. A successful
CMI contains viable organisms at sites to which they have migrated before sensitization was achieved. In the lung,
failure of the host to mount an effective CMI response and tissue repair leads to progressive destruction of lung.
Tumor necrosis factor (TNF)-alpha, reactive oxygen and nitrogen intermediates, and the contents of cytotoxic cells
(granzymes, perforin) may all contribute to the development of caseating necrosis. Caseous necrosis is frequently

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associated with TB but can also be caused by other organisms, including syphilis, histoplasmosis, cryptococcosis,
and coccidioidomycosis. (See related topics.)
Unchecked bacterial growth may lead to hematogenous spread of bacilli to produce disseminated TB. Disseminated
disease with lesions resembling millet seeds has been termed miliary TB. Bacilli can also spread mechanically by
erosion of the caseating lesions into the airways; at this point, the host becomes infectious to others. In the absence
of treatment, death ensues in up to 80 percent of cases [11]. The remaining patients develop chronic disease or
recover. Chronic disease is characterized by repeated episodes of healing by fibrotic changes around the lesions
and tissue breakdown. Complete spontaneous eradication of the bacilli is rare.
Reactivation disease Reactivation TB results from proliferation of a previously latent bacteria seeded at the time
of the primary infection. Among individuals with latent infection and no underlying medical problems, reactivation
disease occurs in approximately 5 to 10 percent of cases during their lifetime [1]. Immunosuppression is clearly
associated with reactivation TB, although it is not clear what specific host factors maintain the infection in a latent
state and what triggers the latent infection to break containment and become active. Immunosuppressive conditions
associated with reactivation TB include:
HIV infection and AIDS
End-stage renal disease
Diabetes mellitus
Malignant lymphoma
Corticosteroid use
Inhibitors of TNF-alpha and its receptor
Diminution in cell-mediated immunity associated with age
Cigarette smoking [5,12,13]
The disease process in reactivation TB tends to be localized (in contrast with primary disease); in general, there is
little regional lymph node involvement and less caseation. The lesion typically occurs at the lung apices, and
disseminated disease is unusual unless the host is severely immunosuppressed.
Prior TB infection, contained as latent tuberculosis, confers protection against subsequent TB disease [14]. One
review evaluating 23 paired cohorts (total more than 19,000 individuals) noted that individuals with latent
tuberculosis had 79 percent lower risk of progressive tuberculosis following reinfection compared with uninfected
individuals [15].
On the other hand, prior TB disease is associated with an increased risk of subsequent TB disease. Studies in both
HIV-uninfected and HIV-infected individuals with one episode of active TB have noted a two- to fourfold increased
risk of a second episode of active TB compared with individuals without prior active disease [15,16]. A study from
South Africa including 612 patients treated for TB documented recurrent disease in 18 percent of cases over a
five-year follow-up period; recurrence occurred after successful treatment in 14 percent of cases [17]. By comparing
DNA fingerprints of the M. tuberculosis isolates from the first and second episodes of TB, the investigators showed
that 77 percent of the recurrences were new infections rather than relapse [17]. The rate of reinfection TB was four
times the rate of new TB.
MICROBIOLOGY M. tuberculosis belongs to the genus Mycobacterium, which includes more than 50 other
species, often collectively referred to as nontuberculous mycobacteria. Tuberculosis (TB) is defined as a disease
caused by members of the M. tuberculosis complex, which includes the tubercle bacillus (M. tuberculosis), M. bovis,
M. africanum, M. microti, M. canetti, M. caprae, and M. pinnipedii [18]. (See "Microbiology of nontuberculous
mycobacteria".)
Cell envelope The cell envelope is a distinguishing feature of the organisms belonging to the genus
Mycobacterium. The mycobacterial cell envelope is composed of a core of three macromolecules covalently linked
to each other (peptidoglycan, arabinogalactan, and mycolic acids) and a lipopolysaccharide, lipoarabinomannan
(LAM), which is thought to be anchored to the plasma membrane [19]. The outermost layer, the mycobacterial outer

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membrane (MOM), consists of a lipid bilayer structure [20].

Mycolic acid, a beta-hydroxy fatty acid, is the major constituent of the cell envelope, accounting for more than 50
percent by weight; this structure defines the genus. Glycolipids are attached to the outside of the envelope layer
through a connection to the mycolic acid layer; proteins are also embedded in this cell wall complex. Glycolipid
components are implicated in "cord formation," whereby tuberculosis bacilli clump together forming a serpiginous
structure seen on microscopy [21].
Staining characteristics The cell wall components give Mycobacterium its characteristic staining properties. The
organism stains positive with Gram stain. The mycolic acid structure confers the ability to resist destaining by acid
alcohol after being stained by certain aniline dyes, leading to the term acid-fast bacillus (AFB).
Microscopy to detect AFB (using Ziehl-Neelsen or Kinyoun stain) is the most commonly used procedure to diagnose
TB in the world, especially in developing countries with limited laboratory capacity. A specimen must contain at least
104 colony forming units (CFU)/mL to yield a positive smear [22]. Microscopy of specimens stained with a
fluorochrome dye (such as auramine O) provides an easier, more efficient, and approximately a 10-fold more
sensitive alternative. However, microscopic detection of mycobacteria does not distinguish M. tuberculosis from
nontuberculous mycobacteria.
Growth characteristics A distinguishing feature of M. tuberculosis is its slow growth rate. In artificial media and
animal tissues, its generation time is about 20 to 24 hours (as opposed to 20 minutes for organisms such as
Escherichia coli).
Isolation in the laboratory Artificial media used to cultivate M. tuberculosis include potato- and egg-based
media, such as Middlebrook 7H10 or 7H11, or albumin in an agar base, such as the Lwenstein-Jensen medium
[23]. A liquid medium, such as Middlebrook 7H9, is used for subcultures and for propagating the bacillus to extract
DNA for molecular diagnostic and strain-typing procedures [24]. Three to four weeks are required to recover the
organism, depending on the initial quantity of organisms in the specimen.
Broth-based culture systems to improve the speed and sensitivity of detection have been developed. The BACTEC
(BD Diagnostics, Sparks, MD) 460 system is based upon Middlebrook 7H12 medium containing 14C palmitic acid
with a mixture of antibiotics (PANTA) to suppress other bacterial growth [25]. The addition of NAP (p-nitro-alphaacetylamino-beta-hydroxypropiophenone) in the medium suppresses growth of other M. tuberculosis complex
organisms, such as M. bovis, but does not differentiate M. tuberculosis from other nontuberculous mycobacteria.
BACTEC 460 has been phased out by its manufacturer.
Bacterial growth is indicated by the detection of 14C released by M. tuberculosis as it metabolizes the palmitic acid.
In AFB smear-positive specimens, the BACTEC system can detect M. tuberculosis in approximately 8 days
(compared with approximately 14 days for smear-negative specimens) [26,27]. However, the high cost of the
equipment and the need for radioactive material that requires disposal exclude its use in most endemic settings.
Other broth-based systems include Septi-Chek AFB (BBL) and the Mycobacterial Growth Indicator Tube (MGIT, BD
Diagnostics) [28,29]. Septi-Chek AFB is a biphasic system comprised of a capped bottle containing modified
Middlebrook 7H9 broth under CO2 and a paddle coated with solid agar, such as Middlebrook 7H11 and LwensteinJensen media [28]. The recovery rate of M. tuberculosis complex from AFB smear-negative specimens by this
procedure is about 15 to 30 percent higher than conventional media, and the average number of days to recovery is
two to five days shorter [28].
The MGIT 960 system used in the United States is based on Middlebrook 7H9 broth containing silicon rubber
impregnated with ruthenium pentahydrate that serves as a fluorescence quenching-oxygen sensor. As oxygen is
consumed by metabolizing bacteria, fluorescence of the liquid growth medium is detected visually. Among smearnegative samples in a study of 1500 clinical specimens, the recovery rate of the M. tuberculosis complex was about
15 percent less by the MGIT system (68 percent) compared with that obtained by the radiometric BACTEC system,
but the mean time to detection was similar (9.9 versus 9.7 days) [29,30]. The lack of need for expensive
instrumentation and radioactive materials renders MGIT widely acceptable and suitable in many laboratories.

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A similar broth-based and colorimetric detection system is the MB/BacT system (bioMrieux, Durham, NC). In this
system, a colorimetric sensor is embedded at the bottom of a bottle, and when carbon dioxide is produced by a
growing microorganism, the sensor changes from dark green to yellow. This change in color is monitored
continuously by a detection device. A systematic review of this system (compared with the BACTEC 460) found that
the MB/BacT system had a sensitivity of 96 to 100 percent and a specificity of 78 to 100 percent [31].
The Versa TREK (Trek Diagnostic Systems, West Lake, Ohio) system is an automated detection system based on
discerning a change in gas pressure (oxygen consumption by a growing microorganism) in a sealed container. The
systematic review comparing Versa TREK with BACTEC-460, found a sensitivity from 82 to 100 percent and a
specificity of 50 to 100 percent [31].
A continuous automated mycobacterial liquid culture system (CAMLiC) has also been described [32]. This system is
more sensitive than Lwenstein-Jensen culture in identifying M. tuberculosis complex organisms (98 versus 85 to 90
percent) with a mean time to recovery of 13.4 days. It has not been compared yet to BACTEC, Septi-Chek AFB, or
MGIT and is not available in the United States.
Identification of the organism Once the organism is isolated, identification is based on morphologic and
biochemical characteristics, although nucleic acid-based detection methods have obviated many of the conventional
tests. M. tuberculosis is identified by its rough, nonpigmented, so-called "corded" colonies on albumin-based agars.
It is typically positive in the niacin test, has a weak catalase activity, which is inactivated at 68C, and reduces nitrate
[23]. (See "Diagnosis of pulmonary tuberculosis in HIV-uninfected patients", section on 'Nucleic acid amplification'.)
The only other major slow-growing Mycobacterium that is niacin testpositive is M. simiae. Although all members of
the mycobacteria species produce niacin (usually undetectable by the niacin test), the differences in the activity of
the enzymes involved in the salvage pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis in M.
tuberculosis determines niacin positivity in M. tuberculosis. Niacin accumulates in M. tuberculosis because
nicotinamidase, which converts nicotinamide to niacin, is several-fold more active, and the enzyme that recycles
niacin to produce NAD is less active than in members of most other mycobacterial species [33].
The niacin, nitrate reductase, and catalase tests are the three biochemical tests most frequently used to distinguish
M. tuberculosis from other mycobacterial species [23]. Tests for pyrazinamidase production as well as susceptibility
to thiophen-2-carboxylic acid hydrazide (TCH) will distinguish M. tuberculosis from M. bovis, another member of the
M. tuberculosis complex. M. bovis does not express pyrazinamidase (or nicotinamidase) and is susceptible to less
than 5 mcg/mL of TCH [23,34]. Clinical isolates of M. tuberculosis lacking pyrazinamidase activity have been
described that contain nucleotide point mutations in the gene (pncA) that encodes pyrazinamidase; these isolates
are resistant to pyrazinamide (PZA), one of the first-line drugs used to treat TB [35]. (See "Epidemiology and
molecular mechanisms of drug-resistant tuberculosis".)
Drug susceptibility tests Drug susceptibility testing is a critical function of the mycobacteriology laboratory,
especially with emergence of increasingly encountered drug-resistant M. tuberculosis isolates. Drug-resistant
tuberculosis refers to M. tuberculosis that is resistant to one of the first-line antituberculosis drugs: isoniazid,
rifampin, pyrazinamide, or ethambutol. Multidrug-resistant tuberculosis (MDR-TB) refers to M. tuberculosis that is
resistant to at least isoniazid and rifampin and possibly additional chemotherapeutic agents. Extensively
drug-resistant TB (XDR-TB) refers to M. tuberculosis resistant to at least isoniazid and rifampin as well as at least
one of three injectable second-line drugs (capreomycin, kanamycin, and amikacin) and a fluoroquinolone.
In addition to the conventional methods to test M. tuberculosis drug susceptibility, methods that rely utilizing
automated systems and polymerase chain reaction (PCR)-based tests have been developed [36,37]. In automated
systems (BACTEC and the others), growth detected by the indicator system in drug-containing broth is interpreted
as resistance to the drug. A colorimetric microplate-based Alamar Blue assay evaluated in Peru for 34 M.
tuberculosis isolates was comparable with the BACTEC drug susceptibility test (88 to 94 percent) [38]. The test is
based on colorimetric determination of an oxidation-reduction indicator dye resazurin [39].
The microscopic observation drug susceptibility (MODS) test is another liquid culture based drug susceptibility test

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based on observation of M. tuberculosis growth in liquid broth medium containing a test drug. Culture aliquots are
examined daily via inverted light microscopy for cording, a characteristic growth pattern of M. tuberculosis but not
most nontuberculous mycobacteria (M. marinum can cord) [40]. Absence of growth or cording indicates susceptibility
to the broth test drug. In an evaluation of 3760 sputum samples using MODS, automated MB/BacT system, and
Lwenstein-Jensen culture, sensitivity was 98, 89, and 84 percent, respectively, and the median time to the test
results was 7, 22, and 68 days, respectively [41]. The agreement between MODS and the reference tests for
susceptibility was high for all standard antituberculosis drugs.
Line-probe assays (such as InnoLiPA and GenoTypeMTBDR) are gene probe assays to detect drug-resistant M.
tuberculosis; these combine hybridization assays and a nucleic acid amplification test, such as PCR. The PCR target
is the M. tuberculosis rpoB gene; mutation of this gene is associated with rifampin resistance, which is used as a
surrogate for multidrug resistance (rifampin monoresistance is uncommon). Meta-analyses of these assays have
noted high sensitivity and specificity for rifampin resistance of culture isolates (>95 percent) [42,43]. Sensitivity
decreases for direct clinical specimens, such as sputum. Results can be obtained the same day if the test is
performed on isolated M. tuberculosis or if the clinical specimen (eg, sputum) contains a large number of the
tubercle bacilli.
The Xpert MTB/RIF assay is an automated nucleic acid amplification test that can rapidly identify both the presence
of M. tuberculosis and rifampin resistance. This assay is recommended by the World Health Organization (WHO) to
be used in place of traditional smear microscopy for diagnosis of drug-resistant TB or TB in HIV-infected patients
[44]; it is approved in the United States by the Food and Drug Administration (FDA) for diagnosis of M. tuberculosis
in sputum samples. (See "Diagnosis of pulmonary tuberculosis in HIV-uninfected patients", section on 'Xpert
MTB/RIF assay'.)
Mycobacteriophages carrying reporter luciferase have been evaluated for the detection and susceptibility testing of
M. tuberculosis. In an evaluation of 50 isolates in Mexico, the overall agreement with the BACTEC method was 98.5
percent [45]. Using the cultured organisms, the median susceptibility turnaround time was two days (10 days with
BACTEC). A PCR-based method using molecular beacons (fluorogenic reporter molecules) is a promising rapid
method to detect drug-resistant strains of M. tuberculosis [46]. However, these methods are not clinically available
due to cost and stability of the reagents.
Another mycobacteriophage-based system relies on the ability of phage to infect M. tuberculosis present in a sputum
sample [47]. In this test, decontaminated sputum samples are incubated with a suspension of target-specific
bacteriophage. The phage replicates within the infected bacilli, if present, and lyses them. The suspension
containing released phages is incubated with fast-growing nonpathogenic mycobacteria (M. smegmatis) helper cells
on an agar plate. The released mycobacteriophages infect, replicate, and lyse these helper cells to form clear zones
(plaques) of cell lysis, which indicate the presence of M. tuberculosis in the original specimen. If this assay is done in
the presence of an antimicrobial agent, the presence of plaques suggests drug resistance of the organism present in
the clinical specimen. A meta-analysis of 13 studies evaluating mycobacteriophage-based tests demonstrated that
the tests had high specificity (83 to 100 percent) but variable sensitivity (21 to 88 percent) compared with culture
[48-50]. These tests are not available in the United States.
Genome The complete genome sequence of M. tuberculosis strain H37Rv has been determined [51,52]. The
following characteristics have been described:
The genome has 4,411,529 base pairs, containing about 4,000 genes, with a G+C content of 65.6 percent.
Consistent with the recognized structure of its cell envelope, many genes are devoted to lipid biosynthesis and
metabolism.
The organism contains lipid and polyketide biosynthetic enzymes that are normally found in mammals and
plants and about 250 enzymes involved in fatty acid degradation.
It has only 11 complete pairs of two-component regulatory systems, as opposed to more than 30 such pairs in
organisms like E. coli.

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Approximately 10 percent of the genes in M. tuberculosis are devoted to the production of two families of glycine-rich
proteins called PE (proline-glutamine motifs) and PPE (proline-proline-glutamine motifs). These genes are
composed of polymorphic GC-rich repetitive sequences (PGRSs) and major polymorphic tandem repeats, which
have served as a basis for strain-typing M. tuberculosis clinical isolates [53,54]. The functions of these families of
proteins are unknown. However, the PE/PGRS genes in M. marinum, the cause of fish and amphibian tuberculosis,
are preferentially expressed inside granulomas and macrophages [55].
PATHOGENESIS A variety of approaches to study M. tuberculosis virulence have been devised, including those
examining M. tuberculosis virulence factors, bacterial factors associated with intracellular survival, and genotypic
differences in the community prevalence of clinical strains.
Virulence factors The following M. tuberculosis products were described as virulence factors prior to the
introduction of molecular biology tools to study M. tuberculosis [56-61]:
Mycolic acid glycolipids and trehalose dimycolate ("cord factor"), which can elicit granuloma formation in animal
tissue
Catalase-peroxidase, which resists the host cell oxidative response
Sulfatides and trehalose dimycolate, which can trigger toxicity in animal models
Lipoarabinomannan (LAM), which can induce cytokines and resist host oxidative stress
These products and their variants are found in many members of the Mycobacterium species, so their specific role in
M. tuberculosis pathogenesis is not clear.
Many additional so-called virulence factors have been identified by molecular biology techniques. These factors are
often defined as virulence factors based on comparison with the wild-type M. tuberculosis strain in which disruption
of these molecules leads to (1) diminished ability of the mutant to attach to or enter mammalian cells in vitro, (2)
decreased growth in vitro in artificial medium or inside mammalian cells, (3) attenuation in an animal infection model,
(4) decreased ability to induce cytokines in macrophages infected ex vivo, and (5) inability to induce changes (eg,
inhibition of cell physiology) in cellular targets outside of itself [62].
One common approach to study virulence phenotype of an intracellular pathogen is to identify bacterial surface
products that mediate uptake of the organism into non-phagocytic cells. The first bacterial product to be identified in
this way is the invasin protein of Yersinia pseudotuberculosis [63]. In the early 1990s, a similar approach was used
to identify a protein (known as mycobacterial cell entry protein, or Mce1A) that conferred upon a nonpathogenic E.
coli, an ability to invade HeLa cells [64]. The significance of M. tuberculosis entering epithelial cells in TB
pathogenesis is not clear; most studies examining M. tuberculosis-mammalian cell interactions focus on professional
phagocytic cells (macrophages and dendritic cells). It should be noted, however, that the initial site of lung infection
by M. tuberculosis is the alveolar air space, which is composed of type I and type II pneumocytes; these are
epithelial cells. Type I cells comprise about 96 percent of the alveolar surface area; type II cells cover about 4
percent of the surface area but comprise 60 percent of all the alveolar epithelial cells [65]. Thus, M. tuberculosis is
most likely to encounter and enter pneumocytes before they are taken up by alveolar macrophages. Little is known
about the in vivo interaction of M. tuberculosis with alveolar epithelial cells.
Mce1A is now known to be encoded by a gene located in a 13-gene operon containing genes encoding integral cell
wall proteins [51]. Disruption of this operon leads to enhanced virulence of this mutant compared with wild type in
mouse models [66,67]. This observation may relate to M. tuberculosis's ability to establish latent infection in vivo
(see below).
There are three homologues of mce1 operon (mce2, mce3, mce4) elsewhere in the chromosome arranged in the
same manner as the mce1 operon. Phylogenomic analyses of the mce operons suggest that they may encode
adenosine triphosphate (ATP) binding cassette (ABC) transporters, which may be involved in lipid importation, and
mce4 may play a role in cholesterol import [68,69]. A functional disruption of a fatty acyl-coenzyme A (CoA)
synthetase gene fadD5 in the mce1 operon caused the mutant to become attenuated in mice and to exhibit
diminished growth in minimum medium supplied with mycolic acid as the only carbon source [70]. This led to a

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hypothesis that live M. tuberculosis may recycle mycolic acids from dying M. tuberculosis inside granulomas for their
long-term persistence in a host [70]. As noted below, lipids are a crucial carbon source of energy in vitro and in vivo.
(See "Immunology of tuberculosis".)
Differences in structure, composition, and metabolism of the cell wall lipid molecules contribute to differences in
clinical outcome in mammalian hosts. In vivo, M. tuberculosis metabolizes lipids rather than carbohydrates [61,71].
This shift occurs via a bypass system (glyoxylate shunt) in the Krebs cycle when carbon substrates for glycolysis
become limited. The enzymes involved are isocitrate lyases 1 and 2 (ICL1/2), which allow the utilization of fatty acids
as a sole carbon source [72,73]. (See "Immunology of tuberculosis".)
The M. tuberculosis cell wall contains three classes of mycolic acids: alpha, keto, and methoxy mycolates. The
relative composition of oxygenated mycolates influences growth of M. tuberculosis inside macrophages and in vivo
[74]. An M. tuberculosis mutant lacking trans cyclopropane rings (cmaA2 mutant) in its methoxy and keto-mycolic
acids becomes hypervirulent in the mouse model of infection [75]. On the other hand, another cyclopropane
synthase gene mutant (pcaA) lacking cis cyclopropane rings in its alpha-mycolates is attenuated [76]. Other lipid
molecules shown to have an effect on innate and adaptive immune response includes lipoglycans, sulfolipids, and
phthiocerol dimycocerosate [61,77-79].
Use of signature-tagged transposon mutagenesis to create M. tuberculosis mutants has identified several candidate
genes associated with virulence in the mouse model; these have included genes encoding protein secretion systems
[80] as well as products involved in lipid biosynthesis [81]. M. tuberculosis secretion systems include:
The ESX-1 system, also known as type VII secretion system involved in the secretion of immunodominant
proteins ESAT-6 and CFP-10, has been shown to promote escape of M. tuberculosis or its products into the
cytoplasm [80,82,83]. This secretion system is located in an M. tuberculosis chromosome locus called the
region of difference (RD-1). RD-1 gene mutants of M. tuberculosis demonstrate macrophage growth
attenuation in mice [84,85]. In addition, the RD-1 locus is absent in all Bacille Calmette-Gurin (BCG) vaccine
strains, which is believed to be the basis for the attenuation of BCG. ESX-1 homologues have been identified in
other pathogenic and nonpathogenic bacteria, and its role in pathogenesis is not fully understood.
Sec secretion system (also called the general secretion pathway) is an essential secretion pathway found in all
bacterial species.
The twin arginine transporter (TAT) translocates across the plasma membrane protein substrates with double
arginine residues at the N-terminus. Its role in pathogenesis in mycobacteria is not certain, but the same
system found in other pathogens, such as Pseudomonas aeruginosa, enterohemorrhagic E. coli, and
Legionella pneumophila, is required for virulence [86-88].
Factors associated with intracellular survival In mice, M. tuberculosis can be observed inside alveolar
macrophages and dendritic cells in the lungs 14 days after aerosol infection [89]. M. tuberculosis enters these cells
after binding to a variety of receptors on these cells, including C-type lectin receptors (mannose receptor, DC-SIGN),
scavenger receptors, and complement receptors [90]. It is believed that the engagement of certain receptors can
determine the intracellular fate of M. tuberculosis.
In addition, M. tuberculosis lipids, including lipoarabinomannan, lipomannans, phosphatidylinositol mannosides, and
a 19-kdal lipoprotein, are considered pathogen-associated molecular pattern (PAMP) molecules recognized by
toll-like receptor (TLR)-2 [91,92]. Engagement of these ligands by TLR-2 on macrophages induces a
proinflammatory response, including the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1b,
and IL-12 [77,93]. TLR-4 may also engage M. tuberculosis PAMPs [94,95]. Different clinical strains of M. tuberculosis
have been shown to induce distinct patterns of proinflammatory response after engaging these receptors on
macrophages, which may determine the clinical outcome of an infection [96].
Once inside the phagosomal compartment, M. tuberculosis may inhibit the maturation of the phagosome.
Phagosomal maturation requires conversion of Rab5 into GTP-bound Rab7 and the generation of
phosphatidylinositol 3-phosphate (PI3P) in the phagosomal membrane [97,98]. M. tuberculosis products can inhibit

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these processes [99,100]. Thus, from the very early phase of infection, M. tuberculosis can initiate control of its
intracellular fate.
Once M. tuberculosis establishes an infection, another important pathogenic feature is the ability of the organism to
establish latent infection, which can then give rise to reactivation disease. Since reactivation tuberculosis (TB) is the
most common form of the disease, bacterial factors associated with latency are considered important virulence
factors.
A nonhuman primate model using cynomolgus macaques is providing novel insights into the host-pathogen
relationship in TB infection and disease in humans [101,102].
An in vitro model has been developed that mimics the physiologic state of M. tuberculosis during latency in vivo
[103]. When M. tuberculosis is grown under microaerophilic conditions, a state called nonreplicating, persistent
(NRP1) is produced and glycine dehydrogenase activity is induced. In contrast, growth under anaerobic conditions
produces a state called NRP2 in which glycine dehydrogenase activity decreases, but the organism still survives as
long as the loss of oxygen occurred slowly and it passed through the NRP1 stage for a period of time. When oxygen
is reintroduced to organisms grown anaerobically, the pathogen goes out of the NRP2 state. Such an in vitro system
could potentially be used to examine differential gene expression and thus to identify bacterial factors specifically
required under these growth conditions.
Bacterial survival in stationary phase growth can be used as an in vitro model for studying intracellular persistence.
A sigma factor gene sigF has been identified in M. tuberculosis [104]. This gene is a homologue of alternate sigma
factor gene (rpoS), which is important for stationary phase survival of E. coli and Salmonella spp. In M. tuberculosis,
sigF is expressed during stationary phase, nitrogen depletion, and cold shock but not during exponential phase
growth.
One study identified at least seven proteins specifically expressed during the stationary phase growth of M.
tuberculosis; the predominant expressed protein was an alpha-crystallin-like heat shock protein (acr) [105]. Acr
transcript was also induced in M. tuberculosis inside macrophages; acr gene replacement by homologous
recombination in M. tuberculosis H37Rv led to impaired growth of the organism inside mouse bone marrowderived
macrophages [106]. Thus, acr appears to be important for intracellular survival and replication. Since alpha-crystallin
heat shock protein is found in a variety of cell types, its specific role in the observed phenotype of M. tuberculosis
needs further elucidation.
Several investigators have reported that isocitrate lyase (icl), an enzyme essential for fatty acid metabolism, is
specifically upregulated during growth of M. tuberculosis inside macrophages [72,107,108]. There are two icl genes
in M. tuberculosis; the predicted proteins are 27 percent identical. In a mouse model of M. tuberculosis infection,
deletion of both icl genes led to complete impairment of intracellular replication and rapid elimination of the double
mutant from the lungs [109].
The above studies suggest that bacterial latency is associated with a hypoxic state in the host. Using a whole
genome microarray, a large number of genes that are induced under defined hypoxic conditions were identified
[110]. One of these genes was found to be a transcriptional regulator involved in the induction of acr, dosR [111].
Whether dosR is essential for M. tuberculosis to establish latent infection or is merely a "housekeeping" stress
response regulator for the bacillus to respond to a hypoxic condition has yet to be determined. The dosR genes are
also present in nontuberculous mycobacteria (NTM) and may contribute to the cross-protection against TB afforded
by prior NTM infection [112].
M. tuberculosis has a particular predilection for the lungs. In immunocompetent mice, virulent strains of M.
tuberculosis grow progressively in the lungs but not in the spleen or liver [113]. Even in severe combined
immunodeficient (SCID) mice, M. bovis BCG (a relatively avirulent [vaccine] strain) grows faster in the lungs than in
other organs [114]. In the mouse model, fewer organisms are required to establish a lung lesion by the inhalation
than by intravenous challenge [113]. None of the other pathogenic Mycobacterium species appears to share this
tissue tropism. The factors associated with M. tuberculosis that facilitate this unique characteristic are unknown.

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Differences in virulence of clinical isolates Genotyping of M. tuberculosis isolates has demonstrated a number
of clades that account for a large proportion of new TB cases in different geographic regions, suggesting that such
strains may be more virulent than others. M. tuberculous lineages have been associated with clinical manifestations
of disease. In one study, for example, East Asian lineage was less likely to be associated with extrapulmonary
tuberculosis than Euro-American, Indo-Oceanic, or East-African Indian lineage [115]. Improved understanding of the
role of MTB lineages may provide insights into pathogenicity, infectiousness, progression from infection to active
disease, and, perhaps, response to treatment.
The W-Beijing family of M. tuberculosis strains has a global distribution and appears to have a selective advantage
facilitating rapid expansion in regions with high background TB incidence [116-118]. The biological reasons for this
observation are not fully understood. These strains have been documented to cause outbreaks involving multidrugresistant organisms, although in some regions the majority of W-Beijing strains remain drug susceptible [119-121].
M. tuberculosis Beijing genotype strains appear capable of withstanding tuberculosis treatment, even in the absence
of drug resistance [122]. W-Beijing strains have been associated with extrathoracic disease and HIV infection,
although it is not clear whether HIV infection has contributed to the emergence of these strains [120,123,124]. In
experimental animal models, these strains were highly virulent and BCG vaccination was not protective [125-127].
Another strain called CB3.3 caused over 10 percent of new TB cases in New York City between 1992 and 1994
[128]. This strain was found to be resistant to reactive nitrogen intermediates (RNI) generated in vitro by acidified
sodium nitrite [128]. A strain called CDC1551, also found to be resistant to RNI and reactive oxygen intermediates
(ROI), caused a large outbreak in a rural area near the Kentucky-Tennessee border in 1994 to 1996 [129,130].
Another strain called PG004, responsible for a large cluster of TB cases in one northern California community, was
not resistant to these effector molecules. Instead, in mice, this strain produced relatively mild lung disease, in which
loosely organized granulomas apparently failed to limit the spread of infection and allowed the escape of M.
tuberculosis into alveolar air spaces [131]. This study suggested that an M. tuberculosis strain that causes mild lung
disease may allow individuals with subclinical disease more time to spread infection in the population. Therefore,
such strains would be overly represented in a community. This demonstrates that the predominance of an M.
tuberculosis strain in a community is not necessarily a marker of enhanced pathogenicity (eg, transmissibility is not
equivalent to virulence).
A large school outbreak in the United Kingdom was caused by an M. tuberculosis strain called CH in 2001. Among
254 newly infected children, 77 developed active disease within a year of exposure [132].
The reasons why some lineages cause large outbreaks of rapidly progressive disease and others cause reactivation
tuberculosis are poorly understood.
The biological "fitness cost" of drug-resistant M. tuberculosis may be influenced by compensatory mutations.
Previously, an experimental model showed that rifampin resistance in M. tuberculosis was associated with
competitive fitness cost and that resistant isolates from patients with prolonged treatment exhibited no fitness cost
[133]. However, subsequently, many clinical multidrug-resistant strains have been shown to have mutations in the
RNA polymerase gene associated with high competitive fitness in vitro and high fitness in vivo [134]. In regions of
the world with a high prevalence of multidrug-resistant (MDR)-TB, up to 30 percent of their MDR isolates had such
mutations [134].
HOST FACTORS
Genetic susceptibility to infection Genetic analysis of sibling pairs has been used to evaluate putative genetic
markers for enhanced susceptibility to tuberculosis (TB) in populations in Africa [135]. Possible markers on
chromosomes 15q and Xq were identified; the investigators speculate that finding a susceptibility gene on an X
chromosome may partially explain the increased incidence of TB in males in some populations.
In a mouse model, a locus on chromosome 19 was found to regulate replication of M. tuberculosis in the lungs of
DBA/2 mice that die rapidly of TB compared with C57BL/6 mice, which are more resistant to infection [136]. In a
second study in a mouse model, a single isoform of the intracellular pathogen resistance 1 gene (Ipr1) was found to

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be responsible for the increased resistance to M. tuberculosis infection [137]. Resistance was characterized by
smaller lung lesions containing fewer macrophages, slower M. tuberculosis growth in macrophages, and death of M.
tuberculosisinfected macrophages by apoptosis rather than necrosis.
The closest human homologue to lpr1 is SP110. A study of families from Guinea-Bissau and the Republic of Guinea
identified three polymorphisms in the SP110 gene that are associated with susceptibility to tuberculosis [138].
Acquired susceptibility to infection Investigators examined the interferon (IFN)-gamma response pathway in
three patients with severe, unexplained nontuberculous mycobacterial disease [139]. In all three patients,
IFN-gamma was undetectable following stimulation of whole blood but was detectable when stimulated in the
absence of the patients' own plasma. An autoantibody against IFN-gamma was isolated from the patients' plasma
and was found to be capable of blocking the upregulation of tumor necrosis factor (TNF)-alpha production in
response to endotoxin in blocking induction of IFN-gammainducible genes and in inhibiting upregulation of human
leukocyte antigen (HLA) class II expression on peripheral blood mononuclear cells (PBMCs). These acquired
defects in the IFN-gamma pathway may explain unusual susceptibilities to intracellular pathogens, including
mycobacteria, in patients without underlying, genetically determined immunologic defects.
All of the host genes associated with TB susceptibility account for a tiny fraction of all tuberculosis cases identified in
the world. The most important host factor that determines TB susceptibility to TB is HIV coinfection, followed by
other immunosuppressive conditions, including cancer, diabetes, and immunosuppressive medications.
Environmental factors, such as crowding, low socioeconomic status, poor access to healthcare, and family history,
also contribute substantially to the incidence of TB worldwide, and these are important to understanding TB
pathogenesis.
SUMMARY
Inhalation of Mycobacterium tuberculosis and deposition in the lungs leads to one of four possible outcomes:
immediate clearance of the organism, primary disease (rapid progression to active disease), latent infection
(with or without subsequent reactivation disease), or reactivation disease (onset of active disease many years
following a period of latent infection). (See 'Natural history of infection' above.)
The cell envelope is a distinguishing feature of the organisms belonging to the genus Mycobacterium. Mycolic
acid is the major constituent of the cell envelope; this structure defines the genus. The mycolic acid structure
confers the ability to resist destaining by acid alcohol after being stained by certain aniline dyes, leading to the
term acid-fast bacillus. (See 'Cell envelope' above.)
Microscopy to detect acid-fast bacillus (using Ziehl-Neelsen or Kinyoun stain) is a commonly used procedure
for the rapid diagnosis of tuberculosis (TB); a specimen must contain at least 104 colony forming units
(CFU)/mL to yield a positive smear. Microscopy of specimens stained with a fluorochrome dye (such as
auramine O provides) is a more sensitive and efficient technique. Microscopic detection of mycobacteria does
not distinguish M. tuberculosis from nontuberculous mycobacteria. (See 'Staining characteristics' above.)
The slow growth rate is a distinguishing feature of M. tuberculosis. In artificial media and animal tissues, the
generation time is about 20 to 24 hours, which means that cultures may take from two to six weeks for
detectable growth, depending on the cultivation systems used for laboratory isolation. (See 'Isolation in the
laboratory' above.)
Once the organism is isolated, identification is based upon morphologic and biochemical characteristics,
although nucleic acidbased detection methods have obviated many of the conventional tests. The niacin,
nitrate reductase, and catalase tests are the three biochemical tests most frequently used to distinguish M.
tuberculosis from other mycobacterial species. (See 'Identification of the organism' above.)
The following virulence factors have been described: mycolic acid glycolipids and trehalose dimycolate (which
can elicit granuloma formation in animal tissue), catalase-peroxidase (which resists the host cell oxidative
response), sulfatides and trehalose dimycolate (which can trigger toxicity in animal models),

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lipoarabinomannan (LAM; which can induce cytokines and resist host oxidative stress), and secreted proteins,
including CFP10 and ESAT-6. Molecular biology techniques have identified many other gene products that may
be involved in the ability of M. tuberculosis to enter cells, resist intracellular killing, establish persistence, and
come out of latency. (See 'Virulence factors' above.)
Epidemiologic studies have revealed a few key M. tuberculosis lineages to be overly represented or clustered
in certain communities. Such occurrences may relate to these strains' distinct biological "fitness" or
transmissibility. (See 'Differences in virulence of clinical isolates' above.)
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Topic 8023 Version 18.0

Disclosures
Disclosures: Lee W Riley, MD Nothing to disclose. C Fordham von Reyn, MD Nothing to disclose. Elinor L Baron, MD, DTMH Nothing to
disclose.
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