Microbiology and pathogenesis of tuberculosis

Author
Lee W Riley, MD
Section Editor
C Fordham von Reyn, MD
Deputy Editor
Elinor L Baron, MD, DTMH
Disclosures: Lee W Riley, MD Nothing to disclose. C Fordham von Reyn, MD Nothing to disclose. Elinor L Baron,
MD, DTMH Employee of UpToDate, Inc.
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All topics are updated as new evidence becomes available and our peer review process is complete.
Literature review current through: Sep 2014. | This topic last updated: Jul 03, 2013.
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 the 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 M. tuberculosis and deposition in the lungs
leads to one of four possible outcomes:
Immediate clearance of the organism
Latent infection
Immediate onset of active disease (primary disease)
Onset of active disease many years following exposure (reactivation disease)
Among individuals with latent infection and no underlying medical problems, reactivation disease
occurs in approximately 5 to 10 percent of cases [1]. The risk of reactivation is markedly increased in
patients with HIV [2]. 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
[3-7]. Among the approximately 10 percent of infected individuals who develop active disease,
approximately one-half of them will do so within the first two to three years following infection; such
individuals are said to develop rapidly progressive or primary disease.
The tubercle bacilli establish infection in the lungs after they are carried in droplets small enough (5 to
10 micron) to reach the alveolar space. If the innate defense system of the host fails to eliminate the
infection, the bacilli proliferate inside alveolar macrophages and eventually kill the cells. The infected
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 the 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 may
accompany initial infection.
The bacilli continue to proliferate until an effective cell-mediated immune (CMI) response develops,
usually two to six weeks after infection. Failure by the host to mount an effective CMI response and
tissue repair leads to progressive destruction of the 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 that characterizes a tuberculous lesion. Caseous
necrosis is frequently 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 lung airways; at this point the
host becomes infectious to others. In the absence of treatment, death ensues in 80 percent of cases
[8]. 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 dormant 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 [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 become
overt. 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
The disease process in reactivation TB tends to be localized (in contrast to primary disease); 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.
It is generally believed that successfully contained latent tuberculosis confers protection against
subsequent TB exposure [9]. 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 [10]. However, successful
treatment for TB may not always confer protection against a subsequent episode of TB. In one study
from South Africa including 612 patients treated for TB, recurrence was observed in 18 percent of
cases over a five-year follow-up period; recurrence occurred after successful treatment in 14 percent
of cases [11]. 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 [11]. The rate of reinfection TB was four times the rate of new TB.

MICROBIOLOGY M. tuberculosis belongs to the genus Mycobacterium that 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
[12]. (See"Microbiology of nontuberculous mycobacteria".)
Cell envelope The cell envelope is a distinguishing feature of the organisms belonging to the
genus Mycobacterium. Unlike gram-negative bacteria, there is no true outer membrane in
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 [13].
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 [14].
Staining characteristics The cell wall components give Mycobacterium its characteristic staining
properties. The organism stains positive with Gram's 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; a specimen must contain at least 10(4) colony forming units (CFU)/mL to
yield a positive smear [15]. Microscopy of specimens stained with a fluorochrome dye (such as
auramine O provides) an easier, more efficient, and 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 base media, such as Middlebrook 7H10 or 7H11, or albumin in an agar base, such as the
Lowenstein-Jensen (LJ) medium [16]. 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 [17]. Three to four weeks are required to recover the organism, depending upon 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 (BC Diagnostics, Sparks, MD) system is based upon Middlebrook 7H12 medium
containing 14C palmitic acid with a mixture of antibiotics (PANTA) to suppress other bacterial growth
[18]. The addition of NAP (p-nitro-alpha-acetylamino-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.
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 eight days (compared to approximately 14 days for smear-negative specimens) [19,20].
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) [21,22]. 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 LJ media [21]. 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 [21].
MGIT 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 to that obtained by
the radiometric BACTEC system, but the mean time to detection was similar (9.9 versus 9.7 days)
[22]. The lack of need for expensive instrumentation and radioactive materials should render MGIT
widely acceptable and suitable in many laboratories.
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 [23].
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 BACTEC460, found a sensitivity from 82 to 100 percent and a specificity of 50 to 100 percent [23].
A continuous automated mycobacterial liquid culture system (CAMLiC) has also been described [24].
This system is more sensitive than LJ 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 upon
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, socalled 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 [16]. (See "Diagnosis of pulmonary
tuberculosis in HIV-negative patients", section on 'Nucleic acid amplification'.)
The only other major slow-growing mycobacterium that is niacin test-positive 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 NAD biosynthesis in M.
tuberculosis determines niacin positivity in M. tuberculosis. Niacin accumulates in M. tuberculosis
because nicotinamidase that 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 [25].
The niacin, nitrate reductase, and catalase tests are the three biochemical tests most frequently used
to distinguish M. tuberculosis from other mycobacterial species [16]. 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 [16,26].
Clinical isolates of M. tuberculosis lacking pyrazinamidase activity have been described which 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 [27]. (See "Epidemiology
and molecular mechanisms of drug-resistant tuberculosis".)
Drug susceptibility tests Drug susceptibility testing is of growing importance with emergence of
increasingly 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 drugresistant TB (XDRTB) 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 PCR-based tests have been developed [28,29]. 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) [30]. The test is based on colorimetric determination of an oxidation-reduction indicator
dye resazurin [31].
The microscopic observation drug susceptibility (MODS) test is another liquid culture based drugsusceptibility test 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 non-tuberculous mycobacteria [32]. 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 [33]. The agreement between MODS and the reference tests for susceptibility was high
for all standard anti-tuberculosis drugs.
Line-probe assays (such as InnoLiPA and GenoTypeMTBDR) are gene probe assays to detect drugresistant 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) [34,35]. 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) contained a large
number of the tubercle bacilli.
The Xpert MTB/RIF is an integrated system that combines sample preparation in a modular cartridge
system and real-time PCR. In 2010 this technique was recommended by the WHO to be used in place
of traditional smear microscopy for diagnosis drug-resistant TB or TB in HIV-infected patients [36].
(See "Diagnosis of pulmonary tuberculosis in HIV-negative patients".)
In various validation studies in both developed and developing countries, this test has been shown to
have a sensitivity of >98 percent in sputum smear-positive TB cases and 75 to 90 percent in smearnegative TB cases [37-39]. The sensitivity in the detection of rifampin-resistant M. tuberculosis
exceeded 97 percent, while specificity ranged 98 to 100 percent in differentiating TB or rifampin-

resistant TB. The test can yield results in less than two hours [37-39]. Here, rifampin resistance is
assessed as a surrogate for multidrug-resistant M. tuberculosis.
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 [40]. 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 drugresistant strains of M. tuberculosis [41]. 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 [42]. 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,
non-pathogenic 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 to culture [43-45].
Genome The complete genome sequence of M. tuberculosis strain H37Rv has been determined
and is annotated (www.sanger.ac.uk/Projects/M_tuberculosis/) [46]. 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.
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 [47,48]. The functions of these families of proteins are unknown. However,
the PE/PGRS genes in Mycobacterium marinum, the cause of fish and amphibian tuberculosis, are
preferentially expressed inside granulomas and macrophages [49].
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 [50-55]:

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 [56].
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 [57].
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
[58]. 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
[59]. 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 located in a 13-gene operon containing genes encoding integral cell wall
proteins [46]. Disruption of this operon leads to enhanced virulence of this mutant compared to wild
type in mouse models [60,61]. 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 ATP binding cassette (ABC) transporters which may be involved in lipid
importation, and mce4 may play a role in cholesterol import [62,63]. 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 [64]. This led to a hypothesis that live M. tuberculosis may recycle mycolic
acids from dying M. tuberculosis inside granulomas for their long-term persistence in a host [64]. As
noted below, lipids are a crucial carbon source of energy in vitro. (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 [55,65]. 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 [66,67].
(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 [68]. 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 [69]. On the other hand, another cyclopropane synthase gene mutant (pcaA) lacking cis
cyclopropane rings in its alpha-mycolates is attenuated [70]. Other lipid molecules shown to have an
effect on innate and adaptive immune response includes lipoglycans, sulfolipids, and phthiocerol
dimycocerosate [55,71-73].
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 [74] as well as products involved in lipid biosynthesis [75]. M.
tuberculosis secretion systems include:
The ESX-1 system, a secretion system involved in the secretion of immunodominant proteins
ESAT-6 and CFP-10, has been shown to promote escape of M. tuberculosis into the cytoplasm
[74,76,77]. This secretion system is encoded by 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 [78,79]. In addition, the RD-1 locus is absent in all 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), which is an essential
secretion pathway found in all bacterial species.
The twin arginine transporter (TAT), which 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, Legionella pneumophila, is required for
virulence [80-82].
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 [83]. 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 [84].
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 2 (TLR-2) [85,86]. Engagement of these ligands by
TLR-2 on macrophages induces a proinflammatory response, including the expression of tumor
necrosis factor (TNF-a), interleukin 6 (IL-6), IL-1b, and IL-12 [71,87]. TLR-4 may also engage M.
tuberculosis PAMPs [88,89]. 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 [90].
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 [91,92]. M.

tuberculosis products can inhibit these processes [93,94]. 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 is the most common form of the disease, bacterial factors associated with
latency are considered important virulence factors.
In the 1950s and 1960s, investigators developed a mouse model to study latency [95-97]. Mice were
intravenously infected with M. tuberculosis and immediately treated with isoniazid (INH)
and pyrazinamide for 12 weeks. Cultures of the animal tissues for up to six weeks following
completion of the treatment were negative, but nearly one-third of the animals developed TB after
about 12 weeks and two-thirds developed disease after about 24 weeks. It is not clear how
representative this model is for latent M. tuberculosis infection in humans. Several variants of this
model known as the Cornell model have been evaluated [98]. The model is highly dependent on the
parameters used to establish latency and each version of the model has its own set of limitations.
An in vitro model has been developed that mimics the physiologic state of M. tuberculosis during
latency in vivo [99]. 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 [100]. 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) [101]. 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 marrow-derived macrophages [102]. 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
[66,103,104]. 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 [105].
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 [106]. One of these genes was found to be a transcriptional regulator
involved in the induction of acr, dosR [107]. 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 non-tuberculous
mycobacteria (NTM) and may contribute to the cross protection against TB afforded by prior NTM
infection [108].
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 [109]. Even in severe
combined immunodeficient (SCID) mice, M. bovis BCG (a relatively avirulent (vaccine) strain) grows
faster in the lungs than in other organs [110]. In the mouse model, fewer organisms are required to
establish a lung lesion by the inhalation than by intravenous challenge [109]. 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.
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 [111]. 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 [112114]. The biological reasons for this observation are not fully understood. These strains have been
documented to cause outbreaks involving multidrug resistant organisms, although in some regions the
majority of W-Beijing strains remain drug susceptible [115-117]. M. tuberculosis Beijing genotype
strains appear capable of withstanding tuberculosis treatment, even in the absence of drug resistance
[118]. 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 [116,119,120]. In
experimental animal models, these strains were highly virulent and BCG vaccination was not
protective [121-123].
Another strain called CB3.3 caused over 10 percent of new TB cases in New York City between 1992
and 1994 [124]. This strain was found to be resistant to reactive nitrogen intermediates (RNI)
generated in vitro by acidified sodium nitrite [124]. 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 1994-96 [125,126].
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 [127]. 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
[128].

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 [129]. 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 [130]. In regions of the world with a high prevalence
of MDR TB, up to 30 percent of their MDR isolates had such mutations [130].
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 [131].
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 to C57BL/6 mice that are more resistant to
infection [132]. In a second study in a mouse model, a single isoform of the intracellular pathogen
resistance 1 gene (Ipr1) was found to be responsible for the increased resistance to M. tuberculosis
infection [133]. Resistance was characterized by smaller lung lesions containing fewer macrophages,
slower M. tuberculosis growth in macrophages, and death of M. tuberculosis-infected 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 [134].
Acquired susceptibility to infection Investigators examined the interferon-gamma (IFN-gamma)
response pathway in three patients with severe, unexplained nontuberculous mycobacterial infection
[135]. 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 IFNgamma was isolated from the patients' plasma and was found to be capable of blocking the
upregulation of TNF-alpha production in response to endotoxin, in blocking induction of IFN-gammainducible genes, and in inhibiting upregulation of 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 co-infection, 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, rapid progression to active disease

(primary disease), or latent infection (with or without subsequent reactivation disease).

(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
10(4) 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 acid-based 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), and lipoarabinomannan (LAM) (which can induce cytokines and resist
host oxidative stress). 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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