Tuberculosis: Natural history, microbiology, and pathogenesis

INTRODUCTION — Mycobacterium tuberculosis causes tuberculosis (TB) and is a leading infectious cause of death in adults worldwide [1]. The human host serves as a natural reservoir for M. tuberculosis. (See "Epidemiology of tuberculosis".)

The microbiology and pathogenesis of TB 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 [2-4]. The risk of reactivation is markedly increased in patients with HIV and other medical conditions [5,6]. 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 TB comes from human autopsy data prior to the era of antituberculosis drugs and from experimental animal models [7-11]. 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 (called Ghon focus) produced by the expansion of the tubercle into the lung parenchyma and lymph node enlargement or calcification together comprise the Ranke complex [12]. 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), which function to eliminate M. tuberculosis, may also contribute to collateral host cell damage and the development of caseating necrosis. Hence, much of TB pathology results from an infected host's proinflammatory immune response to the tubercle bacilli. 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 airways; at this point, the host becomes infectious to others. In the absence of treatment, death ensues in up to 80 percent of cases [13]. 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, it has generally been estimated that the lifetime risk of reactivation disease after an index infection is 5 to 10 percent, with a 5 percent risk in the two to five years following infection and another 5 percent risk over the remaining lifetime [4]. A study from Australia of close contacts of TB cases reported a risk of 11 percent in the five years following infection; the risk rose to 14.5 percent after adjusting for death and loss to follow-up [14].

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

●Chronic and end-stage kidney disease

●Diabetes mellitus

●Malignant lymphoma

●Corticosteroid use

●Inhibitors of TNF-alpha and its receptor

●Diminution in cell-mediated immunity associated with age

●Cigarette smoking [6,15,16]

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 TB, confers some protection against subsequent TB disease [17]. One review evaluating 23 paired cohorts (total more than 19,000 individuals) noted that individuals with latent TB had 79 percent lower risk of progressive TB following reinfection compared with uninfected individuals [18].

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 [18,19]. 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 [20]. 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 [20]. The rate of reinfection TB was four times the rate of new TB. The increased risk of active disease in those with prior TB may be a reflection of the high prevalence of the disease and therefore high transmission frequency in a community with a large number of high-risk hosts.

The term "remote infection" refers to infection that progresses to active TB after ≥2 years of a new infection [21]. Worldwide, particularly in high-burden countries, most clinical TB reflects recently transmitted infection (<2 years prior to development of active disease) [21]. Therefore, it has been proposed that treatment of latent TB infection be prioritized for those who are recently infected.

In low-burden countries, a higher proportion of TB cases occurs due to remote infection among foreign-born residents [22-24]. As an example, in the United States in 2016, a majority of foreign-born persons who developed TB had resided in the United States for more than five years before diagnosis [25]. The percentage varied by place of birth; more than two-thirds of those born in Mexico or the Philippines and about 50 percent of those from India and other countries had resided in the United States for more than five years before diagnosis [25]. It is possible that these individuals became reinfected when they visited their country of origin and developed disease after returning to the United States [21]; however, the likelihood of such reinfections progressing to active disease is low [26,27].

In a systematic review of 312 mathematic modeling studies to assess the incidence of progression from latent infection to active disease, the median annual incidence was found to decrease from 77 to 1.7 cases per 1000 between the 1 and 20 years after infection among individuals with no risk factors [28]. Median cumulative incidence increased from 7.7 to 14.2 percent between 1 and 20 years after infection [28]. However, these modeling estimates varied greatly across studies with 20-year cumulative incidence and were often at odds with empiric observations. Many of these progression studies are limited by the inability to precisely determine the time of exposure that leads to a latent infection.

MICROBIOLOGY — M. tuberculosis belongs to the genus Mycobacterium, which includes more than 50 other species, often collectively referred to as nontuberculous mycobacteria. TB is defined as a disease caused by members of the M. tuberculosis complex, which include the tubercle bacillus (M. tuberculosis), M. bovis, M. africanum, M. microti, M. canetti, M. caprae, M. pinnipedii, and M. orygis [29,30]. (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 [31]. The outermost layer, the mycobacterial outer membrane (MOM), consists of a lipid bilayer structure [32].

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 TB bacilli clump together forming a serpiginous structure seen on microscopy [33].

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 countries with limited laboratory capacity. A specimen must contain at least 10⁴ colony forming units (CFU)/mL to yield a positive smear [34]. 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 Löwenstein-Jensen medium [35]. 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 [36]. 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 exist. The BACTEC 460 system is based upon Middlebrook 7H12 medium containing 14C palmitic acid with a mixture of antibiotics (PANTA) to suppress other bacterial growth [37]. 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. 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) [38,39]. 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) [40,41]. 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 Löwenstein-Jensen media [40]. 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 [40].

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 smear-negative 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) [41,42]. The lack of need for expensive instrumentation and radioactive materials renders MGIT widely acceptable and suitable in many laboratories.

A similar broth-based and colorimetric detection system is the MB/BacT system. 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 [43].

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 68ºC, and reduces nitrate [35]. (See "Diagnosis of pulmonary tuberculosis in adults".)

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 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 [44].

The niacin, nitrate reductase, and catalase tests are the three biochemical tests most frequently used to distinguish M. tuberculosis from other mycobacterial species [35]. 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 [35,45]. 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 [46]. (See "Epidemiology and molecular mechanisms of drug-resistant tuberculosis".)

Genome — The complete genome sequence of M. tuberculosis strain H37Rv was reported in 1998 [47,48]. The following characteristics have been described in this laboratory strain:

●The genome has 4,411,529 base pairs, containing about 4000 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 [49,50]. The functions of these families of proteins are unknown. However, the PE/PGRS genes in M. marinum, the cause of fish and amphibian TB, are preferentially expressed inside granulomas and macrophages [51]. Others have suggested that some PE proteins serve as outer membrane nutrient transport proteins [52], including heme acquisition [53]. These proteins have characteristics of Type VII secretion apparatus-associated substrates and that various subgroups of PE and PPE are likely to exhibit different biological functions [54,55].

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 or survival in an animal model, 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. M. tuberculosis "virulence factors" are often defined as bacterial products whose disruption (based on comparison with wild-type M. tuberculosis) leads to diminished ability of the mutant to attach to or enter mammalian cells in vitro, decreased growth in vitro in artificial medium or inside mammalian cells, attenuation in an animal infection model, decreased ability to induce cytokines associated with disease in macrophages infected ex vivo, inability to evade host immune effector molecules, and inability to induce changes (eg, inhibition of metabolism) in cellular targets outside of itself [62,63]. With M. tuberculosis, these factors may include cell wall lipids, proteins, and their regulatory factors; secreted proteins and their regulators; lipid transporters; metabolic pathways; and other proteins of unknown function [64]. Virulence factors may also be identified epidemiologically by comparing M. tuberculosis strains implicated in community outbreaks to other less frequently represented clinical or laboratory strains [65-71].

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 [72]. 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 [73]. 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 [74]. 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 encoded by a gene located in a 13-gene operon containing genes encoding integral cell wall proteins [47]. Disruption of this operon leads to enhanced virulence of this mutant compared with wild type in mouse models [75,76]. This observation may relate to M. tuberculosis' ability to establish latent infection in vivo (see below).

There are three other 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 [77,78]. 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 [79]. 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 [79]. The mce1 operon has been proposed to encode a transporter system for importing mycolic acids located in the outer leaflet of M. tuberculosis cell wall [64,78,80,81]. Disruption of a 14-gene mce1 operon was shown to lead to changes in more than 400 lipid species in a laboratory strain of M. tuberculosis compared with its wild-type counterpart [82].

The functional role of mce2 and mce3 operons is not yet known, but Pandey and Sassetti have shown that mce4 may serve as a transporter of host cell cholesterol and that it was needed by M. tuberculosis to survive in mice during chronic phase of infection [64]. Another protein, LucA, has been proposed to interact with mce1- and mce4-encoded subunit proteins to facilitate import of fatty acids and cholesterol, respectively [83]. As noted below, lipids are a crucial carbon source of energy in vitro and in vivo and hence for M. tuberculosis' long-term survival inside a host. (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 [84]. In vivo, during chronic phase of infection, M. tuberculosis metabolizes lipids rather than carbohydrates [61,85]. 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 [86,87]. (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 [88]. 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 [89]. On the other hand, another cyclopropane synthase gene mutant (pcaA) lacking cis cyclopropane rings in its alpha-mycolates is attenuated [90]. Other lipid molecules shown to have an effect on innate and adaptive immune response includes lipoglycans, sulfolipids, and phthiocerol dimycocerosate [61,91-93].

Use of signature-tagged transposon mutagenesis to create M. tuberculosis mutants has identified several other candidate genes associated with virulence in the mouse model; these have included genes encoding protein secretion systems [94] as well as products involved in lipid biosynthesis [95]. 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 [94,96,97]. One such product phthiocerol dimycocerosates has been shown to act in concert with the ESX-1 secretion system to facilitate macrophage phagosomal rupture [98]. 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 [99,100]. In addition, the RD-1 locus is absent in all Bacille Calmette-Guérin (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 [101-103].

Factors associated with intracellular and in vivo fate of M. tuberculosis — In mice, M. tuberculosis can be observed inside alveolar macrophages and dendritic cells in the lungs about 14 days after aerosol infection [104]. 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 [105]. 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 [106,107]. 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 [91,108]. TLR-4 may also engage M. tuberculosis PAMPs [109,110]. Different clinical strains of M. tuberculosis have been shown to induce distinct patterns of proinflammatory response after engaging these receptors on macrophages, which determine the clinical outcome of an infection [111]. Recognition of M. tuberculosis by cytosolic sensor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) has been implicated in the induction of type I interferon (IFN) response, but the importance of this response in TB pathogenesis or M. tuberculosis in vivo survival in mice or humans is not established [112-119].

The cyclic di-nucleotides (CDNs) represent another group of PAMPs that serve as targets of the host cytoplasmic surveillance pathway [120,121]. Bacterial CDNs (cyclic di-AMP and cyclic-di-GMP) bind to stimulator of interferon genes (STING) and induce type I interferons (IFN-alpha/beta) which elicit autophagy, an innate immunity mechanism important for controlling intracellular infection [122,123]. M. tuberculosis produces cyclic-di-AMP from ATP or ADP [124]. M. tuberculosis that over-expresses cyclic di-AMP can activate the interferon regulatory factor pathway to generate increased amounts of IFN-beta, which leads to macrophage autophagy [125].

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 [126,127]. M. tuberculosis products can inhibit these processes [128,129]. Thus, from the very early phase of infection, M. tuberculosis can initiate control of its intracellular fate.

Autophagy is a cellular process by which cytoplasmic organelles are targeted for lysosomal degradation inside macrophages. Similarly, autophagy can target intracellular pathogens for clearance [123,130]. Autophagy has been suggested to play a role in M. tuberculosis control [131]. M. tuberculosis co-localizes with autophagy-associated proteins such as ATG5, ATG12, ATG16L1, NDP52, BECN1, and LC3 in phagosomal compartments inside cultured cells [132-135]. However, the importance of autophagy in M. tuberculosis control remains uncertain.

Once M. tuberculosis establishes an infection, an important pathogenic feature is the ability of the organism to establish latent infection, which can then give rise to reactivation disease. Since reactivation TB is the most common form of the disease, bacterial factors associated with latency and transition from latency to active disease are important virulence factors.

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 [136]. Even in severe combined immunodeficient (SCID) mice, M. bovis BCG (a relatively avirulent [vaccine] strain) grows faster in the lungs than in other organs [137]. In the mouse model, fewer organisms are required to establish a lung lesion by the inhalation than by intravenous challenge [136]. 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.

A nonhuman primate model using cynomolgus macaques has provided novel insights into the host-pathogen relationship in TB infection and disease in humans [138,139].

Metabolic factors associated with bacterial survival — An in vitro model has been developed that mimics the physiologic state of M. tuberculosis during latency in vivo [140]. 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 is sometimes used as an in vitro model for studying intracellular persistence. A sigma factor gene sigF has been identified in M. tuberculosis [141]. 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) [142]. 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 [143]. 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 [86,144,145]. 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 [146].

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 [147]. One of these genes was found to be a transcriptional regulator involved in the induction of acr, dosR [148]. 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 [149].

It is not clear whether any of the observations made with in vitro models or non-human animal models of latency are predictive of human latent infection. Thus far, no M. tuberculosis factors associated with progression from a paucibacillary state of latent infection to multibacillary state of disease have been identified. Identification of such factors could lead to targeted intervention (drugs, vaccines) to prevent such transition, which would make a major contribution to TB control.

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 TB than Euro-American, Indo-Oceanic, or East-African Indian lineage [150]. 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 [151-153]. 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 [154-156]. M. tuberculosis Beijing genotype strains appear capable of withstanding TB treatment, even in the absence of drug resistance [157]. 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 [155,158,159]. In experimental animal models, these strains were highly virulent and BCG vaccination was not protective [65,160,161].

Another strain called CB3.3 caused over 10 percent of new TB cases in New York City between 1992 and 1994 [66]. This strain, susceptible to all anti-TB drugs, was found to be resistant to reactive nitrogen intermediates (RNI) generated in vitro by acidified sodium nitrite [66]. 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 [67,162].

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 [68]. This study suggested that an M. tuberculosis strain that causes mild lung disease may allow individuals with subclinical disease to be under recognized in the community and therefore 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 [163]. Formerly called Lineage 15, strain RFL15 (based on IS6110 RFLP typing) belonging to the European-American lineage (based on spoligotyping) has been recognized to be responsible for the largest outbreak of isoniazid-resistant M. tuberculosis in Western Europe that was centered in North London [164,165].

The reasons why some lineages cause large outbreaks of rapidly progressive disease and others cause reactivation TB are poorly understood. The advent of whole-genome sequence (WGS) analysis of bacterial genomes has revealed several sequence differences associated with M. tuberculosis virulence. One such difference is in the so-called region of difference (RD) locus [166,167]. Among members of the M. tuberculosis complex, this region, which is found in M. tuberculosis reference strain H37Rv, is absent in the vaccine strain M. bovis–BCG. Clinical isolates of M. bovis often lack RD4, RD5, RD6, RD7, RD8, RD9, RD10, RD12, and RD13 [64].

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 [168]. 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 [169]. 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 [169].

HOST FACTORS

Genetic susceptibility to infection — Genetic analysis of sibling pairs has been used to evaluate putative genetic markers for enhanced susceptibility to TB in populations in Africa [170]. 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 [171]. 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 [172]. 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 TB [173].

Acquired susceptibility to infection — Investigators examined the interferon (IFN)-gamma response pathway in three patients with severe, unexplained nontuberculous mycobacterial disease [174]. 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-gamma–inducible 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 TB 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.

Progression from latent infection to active disease — Host factors involved in progression from latent infection to active disease have been described in several reviews [175,176]. One large study in Africa attempted to identify host biosignatures predictive of progression from latent TB infection to disease [177]. The investigators prospectively followed South African adolescents 12 to 18 years of age who were infected with M. tuberculosis and compared the blood RNA biosignatures of those who developed TB during the follow-up period with those who did not. The 16 biomarkers identified were found to show a sensitivity and specificity of 54 and 83 percent, respectively, for predicting progression to TB within 12 months preceding TB [177].

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 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), 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, inhibit host cytosolic surveillance, 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.)

●TB pathogenesis can be influenced by host-related characteristics that determine outcomes after an infection with M. tuberculosis. These may involve host genetic factors that enhance susceptibility to infection or progression to disease after exposure. (See 'Host factors' above.)

ACKNOWLEDGMENT — We are saddened by the death of Lee W Riley, MD, who passed away in October 2022. UpToDate acknowledges Dr. Riley's past work as an author for this topic.