M. tuberculosis persistence, latency, and drug tolerance

ARTICLE IN PRESS Tuberculosis (2004) 84, 29–44

Tuberculosis www.elsevierhealth.com/journals/tube

REVIEW

M. tuberculosis persistence, latency, and drug tolerance James E. Gomez*, John D. McKinney Laboratory of Infection Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

KEYWORDS Persistence; Latency; Drug tolerance; Dormancy

Summary The success of Mycobacterium tuberculosis as a pathogen is largely attributable to its ability to persist in host tissues, where drugs that are rapidly bactericidal in vitro require prolonged administration to achieve comparable effects. Latency is a frequent outcome of untreated or incompletely treated M. tuberculosis infection, creating a long-standing reservoir of future disease and contagion. Although the interactions between the bacterium and its host that result in chronic or latent infection are still largely undefined, recent years have seen a resurgence of interest and research activity in this area. Here we review some of the classic studies that have led to our current understanding of M. tuberculosis persistence, and discuss the varied approaches that are now being brought to bear on this important problem. Published by Elsevier Ltd.

Not only does man lack the power to create life but his ability to destroy it, at least at the microbial level, is sharply limitedFWalsh McDermott, 19591

Introduction The invention of antimicrobial chemotherapy in the 20th century was a watershed event in the history of medicine. Thanks to the new ‘‘magic bullets’’, many of the most common and deadly infections that had ravaged the US and Europe for centuries became minor and treatable ailments. The first antimicrobials proved ineffective against tuberculosis (TB), the ‘‘Great White Plague’’ that was responsible for more killing more young adults each year than any other infection. The search for a cure finally culminated, in the mid-1940s, in the discovery of streptomycin by Schatz and Waksman *Corresponding author. Tel.: þ 1-212-327-7082; fax: þ 1-212327-7083. E-mail addresses: [email protected] (J.E. Gomez), [email protected] (J.D. McKinney). 1472-9792/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.tube.2003.08.003

and of para-amino salicylate (PAS) by Lehmann. When the more effective drugs isoniazid (INH) and pyrazinamide (PZA) were introduced in the early 1950s, TB became a treatable disease. As predicted by Ehrlich,2 combination therapy with multiple drugs was widely adopted once it was found that ‘‘under the influence of two different medicines the danger of rendering the parasites immune, which naturally would be a very great obstacle in connection with further treatment, is apparently greatly minimized.’’ In his optimistically entitled book ‘‘The Conquest of Tuberculosis’’, Waksman went so far as to predict that ‘‘the ancient foe of man, known as consumption, the great white plague, tuberculosis, or by whatever other name, is on the way to being reduced to a minor ailment of man. The future appears bright indeed, and the complete eradication of the disease is in sight.’’3 Forty years later, and despite half a century of anti-TB chemotherapy, there are still 8–10 million new cases of active TB each year, and nearly 2 billion individuals are believed to harbor latent TB based on tuberculin skin test (TST) surveys.4 Why

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has this treatable bacterial infection failed to yield to modern medicine? While the full answer to this question is complex, one issue seems clear: the features that enable M. tuberculosis to persist within the tissues of its host have also allowed TB to remain one of the world’s great killers into the 21st century. This problem was anticipated by Ehrlich2 in 1913, in an historic address at the dawn of the chemotherapy era: ‘‘Now that the liability to, and danger of, disease are to a great extent circumscribed y the efforts of chemotherapeutics are directed as far as possible to fill up the gaps left in this ring, more especially to bring healing to diseases in which the natural powers of the organism are insufficient’’. Ninety years later, the ring has not yet been closed in the case of TB, where the ‘‘natural powers’’ of the human immune system are clearly ‘‘insufficient’’ to resolve infection.

Latency, dormancy, and persistence Three terms, latency, persistence, and dormancy, are commonly used in describing M. tuberculosis and TB pathogenesis. Because these terms have not always been used consistently in the literature, they will be defined here as they will be used in this review, with our apologies to those whose ideas differ. Latency was defined by Amberson5 as ‘‘the presence of any tuberculous lesion which fails to produce symptoms of its presence’’. Latency can be achieved through either the early restriction of M. tuberculosis growth in the lungs prior to the onset of TB disease, or the spontaneous resolution of primary TB. Most people exposed to M. tuberculosis mount a vigorous cell-mediated immune response that arrests the progress of the infection, largely limiting it to the initial site of invasion in the lung parenchyma and the local draining lymph nodes (the so-called ‘‘Ghon complex’’).6 The complete elimination of the pathogen, however, is slow and difficult to achieve. Without antibiotic treatment, chronic or latent infection is thought to be the typical outcome of TB infection. Latent TB can reactivate after years or even decades of subclinical persistence, leading to progressive disease and active transmission of the pathogen. Dormancy has been used to describe both TB disease as well as the metabolic state of the tubercle bacillus. TB lesions are described as active or dormant, based on whether the associated pathology is progressing or healing, respectively. Active lesions generally contain easily detectable populations of acid-fast, culturable M. tuberculo-

J.E. Gomez, J.D. McKinney

sis, but the precise bacteriological status of dormant lesions remains unclear despite nearly a century of study and debate. The term dormancy has also become strongly associated with an in vitro model of M. tuberculosis growth under limiting oxygen tension, developed by Wayne.7 It has been suggested that this model may approximate the state of M. tuberculosis surviving in closed, necrotic lesions during clinical latency. It should be emphasized that the model remains speculative, since the location and physiologic state of M. tuberculosis during latency have not yet been firmly established. The word persistence literally means ‘‘continuing steadfastly or obstinately, especially in the face of opposition or adversity’’. As a pathogen, M. tuberculosis manifests its unusual capacity to persist in many ways. On the cellular level, mycobacteria reside within macrophages, cells that typically function to eliminate pathogens and other foreign material from the body. At a more systemic level, M. tuberculosis is able to avoid elimination from the human host despite the development of vigorous cell-mediated immunity. Another less obvious but profoundly important manifestation of M. tuberculosis persistence is the slow rate at which this bacterium is cleared by anti-TB drugs. The six months or more of chemotherapy required to cure TB makes the treatment of this disease an especially formidable challenge to global public health infrastructure, particularly in the developing world.

Persistence ex vivo Long before the discovery of streptomycin and other antimicrobials, M. tuberculosis was known to be an unusually hardy bacterium, both inside and outside the body. In the early 20th century, researchers subjected M. tuberculosis to a barrage of environmental assaults to ascertain which conditions affected the organism’s viability and virulence (8 and references therein). M. tuberculosis proved quite adept at withstanding a wide variety of ex vivo insults, including desiccation, nutrient deprivation, and osmotic shock, but was found to be highly sensitive to direct exposure to sunlight Fan Achilles heel that was later exploited by installation of ultraviolet lights in public spaces such as hospital wards and homeless shelters. It was frequently observed that bacterial samples subjected to environmental affronts retained their virulence for guinea pigs longer that they retained their ability to be subcultured on artificial media.

ARTICLE IN PRESS M. tuberculosis persistence, latency, and drug tolerance

These qualitative observations were probably due in part to the primitive culture media and technology of the time, as well as the exquisite susceptibility of guinea pigs to TB. A more quantitative study of the longevity of tubercle bacilli in culture was carried out by Corper and Cohn, who placed several hundred sealed cultures of various human and bovine isolates in a 371C incubator in 1920 and left them untouched until 1932.8 Of 56 bottles that were subsequently analyzed, 24 yielded culturable organisms, with an estimated survival of 0.01% in the culturable human samples and approximately 1% survival in the bovine isolates. The viability of the cultures was strongly dependent on the pH of the conditioned medium; samples in which the pH had dropped below 6.1 or risen above pH 7.6 failed to yield viable bacteria. Strains that were known to be virulent prior to their placement in the incubator in 1920 retained their virulence upon subculture in 1932. The authors of this remarkable study speculated that the ability of TB to persist in a closed culture vessel for so many years might be connected to longterm persistence during latency, and drew an analogy between their sealed bottles and the healed human lesions thought to contain persistent M. tuberculosis. These observations provided a foundation for the ‘‘Wayne model’’ of TB persistence in oxygen-limited cultures, which has been the focus of renewed interest in recent years.9

Latency Prior to the antibiotic era, TB was considered a lifelong infection: ‘‘Once tuberculous, always tuberculous’’. This clinical adage was recently given a new twist by a molecular epidemiology study in Denmark, which provided the first compelling molecular evidence for the existence of extraordinarily long periods of latency in untreated humans.10 This study examined the case of a Danish man who first developed TB in 1990. When the IS6110 fingerprint of the M. tuberculosis strain isolated from this patient was compared to fingerprints from the national strain collection, the only match was to an isolate dating from 1958Fhis father’s. Despite the common feature of persistence, in the pre-antibiotic era the clinical outcome of infection was clearly variable in different individuals. While some succumbed relatively quickly to a steadily progressive primary infection, others were able to contain the disease. This containment

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was often temporary, and post-primary TB developing after an extended incubation period was commonplace. The reemergence of TB in those who had previously been diagnosed with TB was generally assumed to be due to reactivation of the earlier infection. The introduction of antibiotic therapy substantially reduced the risk of reactivation, further underscoring the inability of many patients to eliminate M. tuberculosis from the tissues without therapeutic intervention. In the early years of chemotherapy, post-therapy relapse rates hovered around 20%Fa substantial improvement from the era when 50–80% of TB cases proved fatal within a few years of diagnosis, but still far from ideal. A major step forward was taken in the 1960s with the introduction of shortcourse combination chemotherapy consisting of INH, rifampin (RIF), and PZA, which further reduced the post-therapy relapse rate to a more acceptable 1–2%.11 Historically, the contribution of exogenous reinfection to the incidence of post-primary TB was largely ignored because it was assumed (on scant evidence) that the first infection would provide substantial protection against secondary infection. Recently, however, it has become clear that exogenous reinfection can be an important source of post-primary TB in high-prevalence areas.11,12 It is currently unclear what proportion of the incident cases of postprimary TB on a global scale is contributed by exogenous reinfection vs. endogenous reactivation. As discussed by Styblo,13 the relative importance of these two sources of postprimary TB in a geographically delimited population is probably determined by the local trajectory of the epidemic. Where TB rates are high and rising, as in much of sub-Saharan Africa, reinfection should become increasingly important; where TB rates are low and falling, as in the US and Western Europe, reactivation should predominate. It is widely assumed that latent TB is due to the incomplete healing of tuberculous lesions, that these smoldering lesions are the site of persistence of dormant tubercle bacilli, and that, under certain circumstances, the standoff between host and parasite shifts in the bacterium’s favor, allowing reactivation. The puzzle is how to reconcile this picture with the fact that the human immune system is clearly capable of mounting a vigorous response against M. tuberculosis. Although locally destructive, this necrotizing response can lead to the extensive fibrosis of tuberculous lesions, caseation of infectious foci, and the eventual calcification of the surrounding tissue.14,15 Is it possible that tubercle bacilli are capable of persisting for years within these apparently

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healed lesions? Or does lympho-hematogenous or bronchogenic spread allow the persistence of tubercle bacilli in other regions of the lung? And finally, do other tissues or cell types also harbor viable bacilli during latent infection? Conclusive answers to these questions are not yet available, although the issues have been addressed by many investigators for the better part of a century (reviewed in 16). Surgical resection of tuberculous lesions was uncommon in the pre-antibiotic era, so early attempts to assess the viability of tubercle bacilli in human lesions were largely limited to necropsy specimens. Lesions obtained from cadavers were frequently shown to contain acid-fast bacilli, sometimes in great numbers. In the mid-1920s, Opie and Aronson collected several hundred necropsy specimens and inoculated this material into guinea pigs.17 They found that homogenates of fibrocaseous lesions of the apex of the lung typically caused TB in guinea pigs, whereas homogenates of caseous encapsulated or calcified lesions seldom did. Interestingly, nearly half of the samples derived from superficially normal lung tissue were found to be infectious for guinea pigs. In contrast, Feldman and Baggenstoss, working at the Mayo Clinic in the late 1930s, found tubercle bacilli in normal lung tissue in only 3 of 51 cases.18 These authors also observed that the Ghon complex, the calcified primary tubercle and draining tracheobronchal lymph node, was almost never infectious for guinea pigs.19 Similarly, Sweaney et al. were unable to recover viable tubercle bacilli from most lesions and the tissue surrounding them.20 Together these data suggested that humans are capable of sterilizing primary lesions in the lung and draining lymph nodes, but that tubercle bacilli may persist more effectively in newer secondary lesions, or perhaps even in essentially normal tissue. A new twist was recently added to this story: more than 70 years after Opie and Aronson’s original studies were published, the presence of IS6110 DNA, an insertion element found in multiple copies in the M. tuberculosis chromosome, was demonstrated in the superficially normal lung tissue of Ethiopians and Mexicans who died of causes other than TB.21 In situ PCR revealed that mycobacterial genomic DNA frequently localized to cells other than macrophages, including lung epithelial cells and Type 2 pneumocytes. This intriguing study raises the possibility that invasion of non-professional phagocytes lacking MHC class II antigenpresenting molecules may be a novel immune evasion strategy of M. tuberculosis; however, the persistence of viable bacilli was not explicitly demonstrated.

J.E. Gomez, J.D. McKinney

Drug tolerance If the fate of bacilli in naturally healed lesions continues to be controversial, the status of tubercle bacilli within patients treated with antimicrobials is even less clear. Following the introduction of TB chemotherapy in the late 1940s, surgical resection of tuberculous lesions became increasingly common and provided material for numerous bacteriological studies. Several reports published in the mid-1950s showed that large numbers of acid-fast bacilli could be observed by microscopy in tubercles resected months after patients on chemotherapy became sputum-negative.22–28 Although M. tuberculosis could be cultured from some of these lesions, most failed to yield culturable bacilli; this was especially true of ‘‘closed lesions’’ lacking patent bronchial communication. However, whether these visible but nonculturable bacteria were truly and irreversibly dead or were instead ‘‘viable but non-culturable’’ remained the subject of vigorous debate.24 A proponent of the latter idea, McDermott speculated that these nonculturable bacteria might be not dead, but merely ‘‘dormant’’, and perhaps capable of being resuscitated under the right conditions. Throughout the 1950s and 1960s, McDermott and colleagues carried out extensive studies in mice to evaluate the fate of M. tuberculosis during and after chemotherapy.29–32 These classic studies, which laid the foundation for our current understanding of the efficacy of antituberculous agents used individually and in combination, led to the development of a mouse model of M. tuberculosis persistence and reactivation (the ‘‘Cornell model’’) still in use today. Work by Hobby and others suggested that inhibitors of mycobacterial growth might be present in resected tissue specimens, and, by careful washing of samples and extended culture times, Hobby’s group was able to substantially increase the rate at which they successfully recovered bacteria from the resected lesions.25 While efforts by others to duplicate these findings were not always successful, Vandiviere and colleagues used extended culturing to obtain even more intriguing results.27 While their 94% success rate in culturing bacilli from the open lesions of drug-treated patients was somewhat high but not exceptional, their observations regarding closed lesions were striking. By extending the incubation period of their cultures from the standard 8 weeks out to 3 to 10 months, they were able to culture bacilli from nine of 22 closed lesions. Seven of these nine isolates were fully drug-sensitive, in contrast to the open cavities, where 37 of 45 isolates were

ARTICLE IN PRESS M. tuberculosis persistence, latency, and drug tolerance

resistant to one or more of the drugs with which the patient was treated. Lack of drug access was presumably not responsible for the survival of M. tuberculosis within the closed lesions, because INH had been shown previously to penetrate all types of lesions in the human lung.33 To explain their observations, the authors postulated that conditions within closed lesionsFsuch as lowered oxygen tension, long-chain fatty acids, lactic acid, and other bacteriostatic agentsFreduced bacterial metabolism and rendered tubercle bacilli refractory to drugs. In this context, it is noteworthy that all of the frontline drugs currently used to treat TB target processes that are involved in cell growth and division, which may explain their poor activity against in vivo bacteria and particularly against bacteria within closed lesions. Despite their success in culturing viable bacilli from closed lesions, Vandiviere and colleagues speculated that ‘‘if this state of dormancy lasted long enough, the normal effect of host resistance y could, with sufficient time, reduce the viable units, finally leading to sterilization of the cavity’’. In a further attempt to characterize the differences between open and closed lesions, Haapanen and coworkers obtained gas samples by needling 20 cavities from the lungs of living TB patients and measuring the concentrations of carbon dioxide and oxygen therein.34 Blocked cavities, where the overall pressure was negative, were enriched for carbon dioxide (10.5% on average, versus 3.5% for open cavities) and partially depleted for oxygen (6.3% on average, versus 17.8% in open cavities). Like Vandiviere and colleagues, these authors also expressed concern that closure of cavities might slow the metabolism of any viable tubercle bacilli remaining inside, thereby interfering with the bactericidal activity of antitubercular agents such as INH and streptomycin. INH and streptomycin were known to be far more effective against actively replicating M. tuberculosis than against non-dividing cells, as demonstrated by Hobby and Lenert in 1957.35 The striking difference in antibiotic susceptibility of actively replicating bacterial cells and non-dividing cells has profound implications for the treatment of TB (see below). These and other classic studies on the bacteriology of resected lesions suggested that the ultimate fate of tubercle bacilli in the treated TB patient might be determined by the interrelated consequences of host defenses and antimicrobial activity. If the environment within the tuberculous lesion is largely bacteriostatic and only slowly bacteriocidal, then the efficacy of antimicrobials may be diminished once the lesions containing the most active bacilli have been successfully treated.

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Eventual eradication of persistent bacteria from the tissues would then require extended chemotherapy either to allow drugs with poor activity against minimally active bacteria to complete their work, or to allow the host to maintain bacteriocidal conditions within the lesion for long enough to kill all remaining bacteria. According to the latter view, the continued presence of antimicrobials would be necessary to kill any bacilli that spread from the infectious focus or resumed active replication in liquefied caseum,15 while contributing only marginally to the late bacteriocidal effects within the lesion. These bacteriocidal conditions would presumably be determined by the immune effector cells present within the healing lesion, as well as the overall character of the lesion (e.g., open vs. closed, necrotic vs. non-necrotic, etc.).

Modeling persistence in vitro The simplest model of M. tuberculosis persistence is the stationary phase culture. The kinetics of replication of M. tuberculosis in the lungs of mice are reminiscent of the organism’s replication kinetics in culture: an initial period of exponential growth is followed by an extended period during which the number of viable bacilli remains stable. Admittedly, the forces that bring about the cessation of growth in vitro (nutrient exhaustion) are not equivalent to those in vivo (acquired immunity, although this may play a role in restricting nutrient availability). Still, the stationary phase culture does provide a simple, inexpensive, and easily manipulated system to analyze the long-term survival of non-replicating cells of M. tuberculosis. Among the more important observations made using axenic stationary phase cultures of M. tuberculosis are those relating to drug susceptibility. In one of the earliest studies of its kind, Hobby and Lenerts demonstrated that when actively growing M. tuberculosis cultures were washed and resuspended in buffered saline (sans carbon source), the resulting cessation of bacterial growth was accompanied by the acquisition of refractoriness to killing by INH and PAS.35 This phenomenon is not unique to the genus Mycobacterium; for example, Hobby had previously observed that stationary phase cultures of E. coli were refractory to killing by penicillin.36 The correlation between the bacterial growth rate and the rate of antibiotic-dependent killing is one manifestation of a more general phenomenon known as antibiotic ‘‘tolerance’’, a term coined by Tomasz in 1970.37 More specifically, slowly

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replicating bacteria are said to exhibit phenotypic tolerance;38 certain mutations can also give rise to genotypic tolerance but there is scant evidence for this form of antibiotic tolerance in M. tuberculosis.39 The mechanisms underlying phenotypic tolerance have been studied in both gram negative and gram positive bacteria. Phenotypic antibiotic tolerance is not heritable, but instead results from exposure to any of a variety of growth-limiting conditions. Among the conditions that result in phenotypic tolerance in non-mycobacterial species are amino acid starvation and acidic pH, conditions which M. tuberculosis may encounter within the host.40 Phenotypic tolerance has been demonstrated to involve the stringent response controlled by the relA gene.38 In response to amino acid deficient conditions, RelA synthesizes tetra- and penta-phosphorylated guanosine (ppGpp and ppGppp, also known as ‘‘magic spot’’). Association of ppGpp with the b-subunit of RNA polymerase results in inhibition of transcription. A rel mutant of M. tuberculosis was recently shown to be unable to sustain chronic infection in mice,41 but no data on the antibiotic tolerance of this mutant is yet available. The early work of Tomasz focused on genotypic tolerance, which is a heritable trait. In a study of Streptococcus pneumoniae mutants that were inhibited, but not killed, by penicillin, Tomasz observed that cell death was not a passive event.37 Mutations in the lytA gene, encoding a cell wall autolysin, allowed S. pneumoniae cultures to survive penicillin treatment. The penicillin-binding proteins in lytA mutants were still inhibited by blactams, but breakdown of the cell wall and lysis did not occur; in the presence of antibiotic, bacterial growth was arrested, but lytA mutants were not killed. If the drug was removed, bacterial replication resumed. There is but one example of genotypically tolerant M. tuberculosis in the literature;39 however, the phenotype of M. tuberculosis isolates described in this report was very modest as compared to pneumococccal lytA mutants, and the genetic basis of tolerance was not investigated. There are no obvious lytA homologs in the M. tuberculosis genome,42 but the LytA protein does not appear to be highly conserved throughout the eubacteria. In fact, little is known about the actual mechanisms of antibiotic-induced death in M. tuberculosis, although studies to better understand this phenomenon are underway in a number of laboratories. Recent research on the response of M. tuberculosis to antibiotics has focused on transcriptional effects of drug exposure.43–46 Also, a model in which unaerated, 100 day-old stationary phase

J.E. Gomez, J.D. McKinney cultures are treated with rifampin (RIF)47 has been developed as a way of studying bacteria that are phenotypically RIF tolerant. As more information becomes available on cell wall synthesis and breakdown, the stringent response, cell division, and cell death in M. tuberculosis, the mechanisms of tolerance that limit the efficacy of TB chemotherapy can be better evaluated. An in vitro model of dormancy that has received much attention recently was developed by Wayne in the 1970s. The conceptual foundation for this model was laid by Wayne in the 1950s, when the controversy surrounding the viability of M. tuberculosis in drug treated lesions was at its peak. Wayne speculated that difficulties in recovering tubercle bacilli from resected lesions might be related to oxygen availability,48 as bacterial adaptation to the reduced oxygen tension within lesions might render them refractory to culture in wellaerated media. Wayne’s attempts to resuscitate bacteria in resected lesions by altering the oxygenation of cultures were unsuccessful.48,49 Later, however, he succeeded in demonstrating that M. tuberculosis was capable of survival under anaerobic conditions in vitro50 and that the bacteria that survived anaerobiosis underwent stage-specific cell cycle arrest.51 In Wayne’s model, unshaken M. tuberculosis cultures were allowed to settle slowly through a self-generated oxygen gradient into the anaerobic conditions present at the bottom of the culture vessel. The gradual transition to a state of anaerobiosis allowed the settling cells to become ‘‘dormant’’ after a transition period that was marked by increased expression of the enzymes isocitrate lyase and glycine dehydrogenase. The latter were proposed to constitute a novel pathway for regeneration of NAD þ in the absence of aerobic respiration.52 The dormant state induced by oxygen deprivation was characterized by a near total shutdown of DNA, RNA, and protein synthesis47,104,51 and a concomitant loss of sensitivity to antibiotics such as INH and RIF, along with increased sensitivity to metronidazole, an antibiotic used to treat anaerobic infections.52 Upon resuscitation of the cultures by oxygenation, the bacteria underwent several rounds of synchronous replication, indicating that cell cycle arrest was stage specific. Although the Wayne model of dormancy is an in vitro phenomenon, it has been suggested that some of the phenotypic alterations observed may be relevant to the in vivo phenomenon of M. tuberculosis latency.53,54 The relevance of the Wayne model has been questioned because treatment of experimentally infected mice with metronidazole failed to affect M. tuberculosis persistence.55,56 However, it can be argued that

ARTICLE IN PRESS M. tuberculosis persistence, latency, and drug tolerance

the mouse model fails to reproduce important forms of tissue pathology, such as caseation necrosis, that have been postulated (but not proven) to influence intra-lesional oxygen tension and bacterial latency.

Modeling persistence in animals The phenomenon of latency has been difficult to reproduce in a tractable small animal model. Apart from non-human primates,57 which are prohibitively expensive and impractical for general use, rabbits provide the closest facsimile of human tuberculosis in terms of tissue pathology and disease progression. Half a century ago, Lurie bred resistant and susceptible strains of rabbits, and demonstrated that the differential outcome of TB infections in these strains was determined by early events prior to the emergence of a specific T-cell response, as well as late events involving acquired cell-mediated immunity.58 Both sensitive and resistant rabbits were able to curtail bacterial replication once a specific T-cell response was established, and to maintain a stable census in the lungs for many months thereafter. The longterm maintenance of a stable bacterial load in the lungs is a common characteristic of animal models of TB, including guinea pigs and mice as well as rabbits. However, the consequences of M. tuberculosis persistence are strikingly different in terms of tissue pathology and disease progression. As described by Lurie, the pathology that develops in persistently infected rabbits includes the softening and liquefaction of caseous tubercles leading to cavitationFa hallmark of human tuberculosis that is not reproduced in the guinea pig or mouse models. This makes the rabbit a uniquely valuable small animal model to study the persistence of M. tuberculosis in cavitary lesions. Despite these advantages, the rabbit model is not widely used today. This is due in part to economic and logistical considerations, but also to a long history of research focused on the immunology and chemotherapy of infectious diseases in the mouse. As mentioned already, infected mice are able to arrest bacterial replication in the lungs within a few weeks of exposure, depending on the dose and the route of inoculation. Low dose aerosol infection, and moderate doses of M. tuberculosis inoculated intravenously, can be used to reproducibly achieve peak bacterial loads in the lungs of mice between 104 and 107 organisms.59 Resistant strains of mice, such as the C57BL/6 strain,60 are capable of surviving for long periods of time with

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such loads, during which time they show slowly progressive pathology in the lungs but few signs of overt disease. The early arrest of bacterial replication and the continued stability of the lung census are dependent upon intact immunity, which makes this model attractive for the study of both host factors needed for controlling infection as well as bacterial genes necessary for persistence within an immune host. In this regard, much has been learned about host immunity through studies in mice. The cytokines IFN-g61,62 and TNF-a63 have been shown to be essential for both the arrest and continued control of M. tuberculosis replication. These cytokines are involved in the activation of macrophages and the induction of inducible nitric oxide synthase (iNOS or NOS2), which has been shown to be essential in mice for protection against TB.64 Neutralization of either TNF-a65 or NOS266 during the persistent phase of infection leads to a resumption of bacterial replication, underscoring their importance in the long-term control of M. tuberculosis. Clinical evidence suggests that TNF-a may also be essential for maintenance of TB latency in humans, because treatment of latently infected individuals with infliximab, a TNF-a blocker, has been associated with increased risk of reactivation.67 Both CD4 þ T cells68 and CD8 þ T cells69 have been shown to have important roles in the sustained arrest of M. tuberculosis in chronically infected mice. The increased risk of reactivation in latently infected individuals who become coinfected with HIV suggests that CD4 þ T cells are also important for maintenance of latency in humans. Most of our current knowledge regarding immune mechanisms involved in protection and pathogenesis in TB derives from studies in the mouse model. However, the mouse is generally considered to be a poor model for TB latency in humans because the bacterial load in chronically infected mice is relatively high, tissue destruction is progressive, and the outcome is invariably fatal. In contrast, humans with latent TB show low bacterial loads on autopsy, with the majority of lesions apparently sterilized; most importantly, the pathological process is arrested in most individuals, with disease progression occurring in only a small minority. While it seems clear that mice are less able than humans to eliminate M. tuberculosis from the lungs, their ability to restrict bacterial expansion for an extended duration provides a system in which the standoff between host and pathogen can be analyzed. Two models can be proposed to account for this stalemate. In one, a balance between continued bacterial replication and host killing may be achieved at the onset of acquired

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immunity. This model is supported by evidence that the granulomas found in another TB animal model, Mycobacterium marinum infection of frogs, are dynamic structures that may allow continued M. marinum replication.70 Alternately, acquired immunity may drive M. tuberculosis into an ‘‘in vivo stationary phase’’, a state in which little or no cell division occurs. This model is supported by a classic study by Rees and Hart.71 They hypothesized that continuous bacterial turnover due to balanced growth and death would lead to an accumulation of bacterial bodies, since they and others had shown that the corpses of heat- or drug-killed M. tuberculosis were quite stable in mouse lungs. Because they observed stable numbers of acid-fast bacilli in the lungs during persistent infection of mice, they concluded that there was little turnover in the M. tuberculosis population. This conclusion was supported by a concomitant study on the heat resistance of M. tuberculosis derived from mouse lungs, which demonstrated that in vivo bacteria, like stationary phase bacteria grown in vitro, were better able to tolerate exposure to 531C.72 The resistance of in vivo bacteria to thermal stress was particularly marked when bacteria were derived from chronically infected rather than acutely infected mice. While direct measurements of M. tuberculosis cell division rates in the lungs of mice have not been carried out, the available evidence would seem to indicate that replication rates are slowed or halted in response to acquired immunity. Another mouse model of TB latency is based on the reduction, but not elimination, of M. tuberculosis populations in the organs of mice via antimicrobial therapy. The ‘‘Cornell model’’, as it is commonly known, was developed by McDermott and colleagues at Cornell University in the 1950s. The contemporaneous debate over the viability of microscopically detectable but nonculturable bacilli found in tissues resected from drug-treated patients led the Cornell group to examine the effects of antimicrobials on M. tuberculosis in the lungs and spleens of mice. They examined the efficacy of four anti-TB agents, including all of the drugs that were in use clinically, alone and in various combinations.30 As assessed by quantitative analysis of plated lung homogenates, many of the antibiotics studied were severely limited in their ability to sustain their antimicrobial activity after only a few weeks of administration. The bacterial census in the lungs and spleen dropped during the initial weeks of treatment, but tended to stabilize over time, particularly in the spleen. These observations were not the result of the emergence of drug resistant mutants, since the populations of surviving bacteria were typically 499% sensitive.

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Even when the drug dosage was increased or multiple drugs were used in combination, a plateau was typically reached after which no additional killing could be achieved. McDermott used the term ‘‘persisters’’ to designate the long-term, drugsensitive survivors of drug therapy and further suggested that ‘‘This capacity of drug-susceptible organisms to survive drug attack when subsisting in an animal body may be designated as microbial persistence’’.73 He also acknowledged the importance of the problem: ‘‘In clinical practice this phenomenon obviously has to do with the posttreatment ‘carrier state’ and with post-treatment relapse. In short, it is this phenomenon which is responsible for our inability to eradicate an infection from a person or a community by the use of drugs.’’ The Cornell investigators found that the only drug that would consistently reduce the bacterial load to undetectable levels in both lungs and spleens was PZA, and only when it was given for a minimum of 12 weeks in combination with a companion drug such as INH.29 Later work by a number of investigators demonstrated that RIF also has potent sterilizing power.74 Despite the initial appearance of success, further investigations established that the ‘‘sterile state’’ that was apparently achieved with INH and PZA was problematic. When mice were sacrificed and examined after 12 weeks of INH and PZA, they appeared devoid of viable M. tuberculosis.31,32 No bacteria could be cultured from these mice, despite efforts that included plating every organ in its entirety, and then homogenizing the remaining flesh and bones and plating that material. Likewise, it proved impossible to transfer the infection to highly susceptible guinea pigs using extracts from these tissues. Yet it became clear that the drugs had not in fact eradicated infection, but merely rendered it ‘‘latent’’ in the sense that ‘‘the presence of the infection cannot be demonstrated by any of the available methods and the fact that it is present can only be detected in retrospect by the appearance of relapse’’.73 When treated mice were maintained for several months without further intervention, the majority began to show spontaneous bacteriologic relapseFi.e., reappearance of culturable bacilli in the tissues. Furthermore, the rate of relapse could be accelerated and increased to 100% of treated animals by administration of high-dose corticosteroids. In keeping with McDermott’s definition of ‘‘persisters’’, the bacilli cultured from the relapsing animals remained fully sensitive to INH and PZA, indicating they were not merely drug-resistant mutants selected during therapy. How had these bacilli ‘‘vanished’’, then

ARTICLE IN PRESS M. tuberculosis persistence, latency, and drug tolerance

reappeared? And what implications did this phenomenon have for the treatment of human TB? One clear implication is the existence of a subpopulation of bacteria in vivo that are phenotypically tolerant to the effects of antibiotics. The question that arises is, what are the conditions in vivo that trigger the tolerant state? Pathophysiologic studies on human lesions led to the hypothesis that the development of potentially bacteriostatic tissue environmentsFe.g., due to reduced oxygen tension, nutrient limitation, or acidic pHFcould result in the acquisition of drug tolerance by non-replicating bacteria. It is important to point out, however, that the forms of tissue damage proposed to contribute to bacteriostasis in human lung lesions seem to differ in important respects from the pathology of tuberculous lesions in mice. The granulomas that develop in human TB are highly organized structures, containing macrophages and giant cells at their center, surrounded by a rim of lymphocytes at the periphery. The necrotic death of cells at the center of these lesions leads to an accumulation of caseous material that may eventually liquefy and cavitate. In contrast, mouse granulomas comprise a looser accumulation of macrophages, neutrophils, and lymphocytes, and caseation necrosis is less common and occurs more slowly.75 Yet, despite the differences in TB tissue pathology in mice and humans, it is clear that M. tuberculosis drug tolerance develops in both host species. Further evidence that in vivo drug tolerance is not strictly dependent on tissue pathology was suggested by Cornell model experiments in which chemotherapy with INH and PZA was initiated immediately after mice were infected.29,30 Despite the absence of accumulated tissue damage, the rate of bacterial killing was very slow and posttherapy relapse was not prevented even if drugs were administered for 12 weeks. Conversely, INH and PZA were still effective when initiation of treatment was delayed until 8 weeks post-infection, although the rate of killing was further reduced as compared to therapy initiated during the acute phase of infection.31,32 Together, these observations would seem to argue against the notion that M. tuberculosis drug tolerance in the Cornell mouse model is linked to the development of host tissue pathology. If tissue pathology does not account for M. tuberculosis drug tolerance in the Cornell model, then what might? While mycobacterial growth inhibition by acquired immunity appears to reduce the initial rate of drug-induced killing in the mouse, it does not prevent the drugs from working altogether. Nonetheless, the Cornell studies

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showed that any drug administered alone, or combinations of drugs not including INH and PZA, became less effective with time. At least two alternative explanations can be envisaged. In the first scenario, chemotherapy would rapidly kill all but a small, highly tolerant subpopulation of bacteria (‘‘persisters’’), which would still be present and capable of reactivating after 12 weeks of treatment. In the second scenario, the slow killing of a moderately but more uniformly tolerant population of organisms during 12 weeks of INH and PZA would be insufficient to achieve complete sterilization by 12 weeks, leaving a small number of surviving bacteria. In either case, but for very different reasons, the surviving organisms would be in a physiologic state that would render them nonculturable and noninfectious to guinea pigs, although capable of reactivating in situ. Either model is consistent with observations of Grosset and colleagues, using fluctuation analysis with genetically marked strains, that the number of organisms reactivating after drug-induced latency is very small, probably less than 10.76 The basic distinction between the two scenarios that have been proposed to account for the difficulty of eradicating TB with drugs is whether a ‘‘special’’ subpopulation of highly drug tolerant bacteria does (first model) or does not (second model) exist in vivo. For the better part of a century, many investigators have proposed that alternate forms of M. tuberculosis cells (e.g., protoplasts, L-forms, or spores)54,77 may go undetected in vivo, such alternate forms being illsuited for growth in culture or transfer to guinea pigs. As yet, however, no compelling evidence has been adduced for the existence of radically altered forms of tubercle bacilli, nor for a role of such altered forms in drug tolerance. Indeed, the kinetics of drug-induced killing in the mouse would seem to support the second model, in which the entire bacterial population is relatively, but not completely, refractory to killing. In this regard, it is noteworthy that, in the Cornell studies, prolongation of INH/PZA treatment to 26 weeks did, apparently, result in complete sterilizationFi.e., the treated animals did not relapse even when severely immune suppressed. This would seem to indicate that the residual bacterial population after 12 weeks of therapy does not consist of nonculturable ‘‘alternate forms’’ that are completely indifferent to drugs. Perhaps the inability to culture organisms from the tissues after prolonged chemotherapy is simply due to accumulated druginduced damage rendering the organisms too fragile to survive the shock of passage to a new environment.

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Since drug tolerance is an in vivo phenomenon, it will be important to ascertain what aspects of the tissue environment are responsible. Unfortunately, this remains a very murky area of mycobacteriology, despite its manifest importance. One important point, at least, is clear: from the very earliest stages of infection, the alterations in bacterial metabolism and physiology that accompany the shift from growth in culture to growth in the lung appear to affect drug activity. This cannot be due solely to alteration of the overall growth rate: during the initial weeks of infection, M. tuberculosis replicates as rapidly in the lungs of mice as in artificial medium, yet drug-dependent kill rates in the two conditions are vastly different. For example, exposure to INH and RIF kills 99.9% of the cells in a culture of M. tuberculosis within 48 h, yet comparable killing with these drugs in vivo requires 3 to 4 weeks. The concentration of antibiotics achieved in the tissues is not likely to account entirely for the reduced drug sensitivity of M. tuberculosis in vivo. Drugs like INH seem to be capable of accumulating to very high levels within TB lesions.33 Thus, it is not surprising that the slow rate of killing in vivo cannot be overcome by simply increasing the drug dosage.78 Why, then, are drugs like INH, which are rapidly bacteriocidal in vitro, so poorly effective in vivo? A definitive answer to this question remains elusive, but an improved understanding of the physiology of M. tuberculosis whilst living in the lungs should provide clues. Recently, the ‘‘Cornell model’’ has been resurrected as a tool for studying various aspects of M. tuberculosis persistence. The original model and its various adaptations are clearly useful for the study of antibiotic effects, particularly their activity against ‘‘persisters’’. The late effects of rifapentine,79 metronidazole,56 moxifloxacin80 and other drugs have been evaluated in various permutations of the Cornell model. The model has also been used to study the immunological control of persistent M. tuberculosis infection,81 (reviewed in 82). The difficulty in reliably achieving the ‘‘sterile state’’ and consistently reviving the infection, in conjunction with the uncertain relationship between druginduced latency and the immunity-driven latency seen in humans, implies that experiments using the Cornell model need to be designed and interpreted with care.81

Recent progress During the past decade, the development of molecular-genetic tools for the analysis of M.

J.E. Gomez, J.D. McKinney

tuberculosis has significantly advanced our ability to study the in vivo biology of M. tuberculosis.83 Genetic and gene expression-based studies have led to the identification of genes that appear to be involved in the adaptation of M. tuberculosis to life in the lungs. The mouse continues to be the most frequently used model for studies of M. tuberculosis pathogenesis, although there has been a renewal of interest in other models, including guinea pigs, rabbits, and non-human primates. Most importantly, new technologies are making it possible, for the first time, to analyze M. tuberculosis physiology directly in tissues obtained from infected humans.84,105 A growing number of genes appear to have their most important role during the late or chronic phases of infection. The ‘‘persistence factors’’ encoded by these genes may offer particularly attractive targets for drug development, with the possibility of shortening the required treatment time significantly.85 One of the first persistence factors identified affected an unusual behavior of M. tuberculosis known as ‘‘cording’’Fi.e., formation of ropelike tangles of laterally associated bacilliFwhich has long been thought to be associated with bacterial virulence.86 The cording phenotype was shown to depend on the pcaA gene product, an enzyme responsible for the cyclopropanation of a-mycolates, which are long chain aalkyl b-hydroxy fatty acids that comprise a major constituent of the mycobacterial cell wall. Disruption of pcaA resulted in a slight enhancement of bacterial replication in mice during the acute phase of infection as well as a later defect in persistence. The rather modest reduction in the bacterial load in the lung at late stages of infection was associated with a seemingly disproportionate impact on the survival of the infected mice. This may have been attributable to the profoundly altered cellular infiltrate and granuloma organization in mice infected with pcaA mutant bacteria lacking proximally cyclopropanated mycolic acids. The role of M. tuberculosis cell wall components in modulating host immunity has been studied intensively, and the cell wall associated molecule pthiocerol dimycocerosic acid (PDIM) has recently been shown to be important for bacterial replication in mouse lungs at early stages of infection.87,106 The pcaA mutant demonstrates that cell wall components may modulate the host response in a stage-specific manner. There has recently been a resurgence of interest in the metabolism of M. tuberculosis during life in the lung. The first observations that ‘‘in vivo grown’’ bacteria were metabolically distinct from bacteria grown in vitro were reported in 1956 by

ARTICLE IN PRESS M. tuberculosis persistence, latency, and drug tolerance

Segal and Bloch. These authors demonstrated that tubercle bacilli isolated from mouse lungs showed a marked enhancement of enzymatic activities related to the breakdown and utilization of fatty acids as a source of carbon and energy.88 Among the pathways required for utilization of fatty acids is the glyoxylate cycle, an anaplerotic pathway that is present in many bacteria but absent in vertebrates. Recently, the icl1 gene encoding isocitrate lyase, one of the enzymes of the glyoxylate cycle, was shown to be essential for late-stage persistence of M. tuberculosis in the mouse model.89 Disruption of the M. tuberculosis icl1 gene had no effect on bacterial growth in vitro or during the acute phase of infection in mice. However, coincident with the onset of cell-mediated immunity, the icl1 mutant was slowly but progressively eliminated from the lungs. This stage-specific persistence defect resulted in a profound attenuation of virulenceFi.e., survival of animals infected with the icl1 mutant was greatly extended as compared to animals infected with wild-type bacteria. The icl1 mutant phenotype was largely reversed in mice deficient in IFN-g; underscoring the key role of the host immune response in driving the dependence of M. tuberculosis on this metabolic pathway. Persistence of M. tuberculosis in vivo presumably involves mechanisms for evading the onslaught of the host immune response. A fascinating example of this interplay may be provided by a recent study in which deletion of the M. tuberculosis hspR gene, encoding a transcription factor that represses expression of the Hsp70 heat shock protein, resulted in a delayed defect in bacterial survival.90 The uncontrolled expression of Hsp70 in M. tuberculosis had no discernable impact on growth in vitro, nor during the acute phase of infection in mice. At later stages of infection, however, the bacterial load was dramatically reduced in mice infected with the hspR mutant bacteria as compared to mice infected with wild-type bacteria. An intriguing possibility is that the uncontrolled expression of the highly immunogenic Hsp70 protein, or a general enhancement of protein (antigen) secretion due to overproduction of this chaperonin, may have resulted in an enhancement of the host immune response and more effective control of infection. Consistent with this idea, splenocytes from mice infected with hspR mutant bacteria contained a twofold higher frequency of Hsp70specific, IFN-g-secreting cells as compared to splenocytes derived from mice infected with wildtype bacteria. The transition from the acute to the chronic phase of infection, accompanied by the shift from exponential bacterial growth to stationary phase

39

persistence, is likely to involve profound alterations in M. tuberculosis gene expression. Sequencing of the M. tuberculosis genome revealed a large repertoire of putative transcriptional regulatory proteins, including 13 sigma factors, 11 complete two-component regulatory systems, and a multitude of additional uncharacterized transcription factors.42 Emerging evidence suggests that some of these regulatory factors are required for the induction and repression of persistence genes. Two-component regulatory systems, consisting of a ‘‘sensor kinase’’ and a ‘‘response regulator’’ that binds to promoter regions to regulate transcription, are involved in the responses to a diversity of environmental stimuli in eubacteria. In M. tuberculosis, disruption of the Rv0981 gene encoding the MprA response regulator was recently shown to impair persistence in the organs of chronically infected mice, with the deficiency being most pronounced in the spleens.91 Alternative sigma ðsÞ factors bind to the core RNA polymerase ða2bb0 Þ and direct it to promoters that are not recognized by the polymerase when it is programmed by the ‘‘housekeeping’’ sigma factor ðsA Þ: In M. tuberculosis, a key role for alternative sigma factors in pathogenesis is emerging from studies in a number of laboratories, and there is some evidence that certain of the alternative sigma factors may have their most important role at later stages of infection.92 Intriguingly, disruption of the M. tuberculosis sigH gene was shown to result in profound attenuation of virulence in mice while having no discernable impact on the bacterial load in the tissues at any stage of infection.93 Likewise, disruption of the M. tuberculosis whiB3 gene, encoding a putative transcriptional regulator, led to prolonged survival of infected mice without affecting bacterial numbers.94 In both cases, mice infected with the mutant bacteria (sigH or whiB3) showed a dramatically altered inflammatory process in the lungs as compared to mice infected with wild-type bacteria. These elegant studies underscore the importance of elucidating the hostpathogen interactions that influence the most important outcome of infectionFdeath or survivalFindependently of the rather crude metric of bacterial numbers in the tissues. An important goal for future studies will be the identification of the key genes controlled by these and other transcriptional regulators and the elucidation of their specific roles in pathogenesis. This is a very active area of TB research and it is anticipated that the key regulatory processes and gene networks that control M. tuberculosis growth, persistence, and pathogenesis at different stages of infection will soon be revealed.

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J.E. Gomez, J.D. McKinney

What next?

1.87 million TB deaths

During the past decade, there has been a resurgence of interest in the phenomenon of M. tuberculosis persistence. In the United States, this has been due in part to the recognition that the demographics of TB are shifting.95 In 1990, threequarters of all TB cases in the United States were among the US-born; a decade later, the numbers of cases among the US-born and foreign-born were roughly equal. Most significantly, from 1990 to 2000, there was a marked fall in the absolute number of TB cases among the US-born; in contrast, during the same period, the absolute number of cases among the foreign-born actually rose, despite increased investment in TB control during the 1990s. These statistics underscore the unusual difficulty of controlling a persistent infection like TB via conventional public health measures, particularly in the context of an increasingly global society like ours in which the far side of the globe is just a plane ride away. Rising rates of immigration to the US from high-incidence countries means that more and more cases of latent TB will enter the country in the years to come. Reactivation after arrival will continue to generate new cases of TB and new foci for transmission within the US. The tools that are currently available to combat this threat to public healthFessentially, the tuberculin skin test and isoniazid chemoprophylaxisFare antiquated and woefully inadequate. In recognition of this unmet need, a recent report from the Institute of Medicine warned that ‘‘Tuberculosis elimination is not possible with the tools that are currently available. The first priority for research is development of an understanding of latent infection’’.96 The fruit of that research should also benefit the world’s poorest countries, which bear the brunt of the global TB epidemic as well as the burgeoning HIV-AIDS epidemic, whose full impact has yet to be felt. HIV infection is the most important single risk factor known for reactivation of latent TB.97 In 2000, nearly half of all TB deaths in Africa were HIV-associated;98 many of these cases were presumably due to reactivation of infections acquired in childhood or adolescence. No effective and practicable intervention is available for large-scale preventive therapy of latent infections in resourcelimited environments. It is probably fair to say that intervention against latent TB is currently not a significant component of current global TB control programs. Given the scale of the problem (Fig. 1), it is difficult to see how TB can be effectively controlled without effective, affordable, and practicable interventions against latent TB infection.

16.2 million TB cases 1.86 billion TST positive 3.94 billion TST negative

Figure 1 TB infection vs disease.4 (’) 1.87 million TB deaths; (N) 16.2 million TB cases; (M) 1.86 billion TST positive; (&) 3.94 billion TST negative.

TB control programs in rich and poor countries alike would benefit enormously from four types of new interventions targeting latent TB. First, an improved diagnostic test for latent TB infection to replace the tuberculin skin test (TST), which lacks sensitivity and specificity. In particular, a diagnostic test that can reliably distinguish between latent TB infection and BCG vaccination will be essential for use in countries where childhood BCG vaccination is routine; significant progress has been made recently towards development of such a discriminatory tool.99 Second, a test to identify those individuals with latent TB who are at risk for future reactivation. Since B90% of latently infected individuals never develop active TB, it would be very useful if the 10% of individuals who will could be identified before reactivation occurs so that resources (e.g., prophylactic therapy) could be focused on them. Third, improved chemoprophylactic therapy for latent TB to prevent reactivation. The regimen currently recommended by the US Centers for Disease ControlF9 months of daily INH monotherapy100Fis impractical for application on a large scale, particularly in resource-limited countries. It is important to note that INH prophylaxis for latent TB infection is not associated with increased risk of acquired INH resistance, implying that the bacterial load must be very small, i.e., less than 106 organisms. Surely it should be possible to devise new drugs that will eliminate such a small population of organisms more effectively and rapidly. An analogous example is provided by the recent introduction of effective single-dose combination therapy (RIF, ofloxacin, minocycline) for singlelesion pauci-bacillary leprosy, caused by the related organism Mycobacterium leprae.101 A comparable regimenFeffective, affordable, safe, and ultra-short courseFfor treatment of latent TB

ARTICLE IN PRESS M. tuberculosis persistence, latency, and drug tolerance

infection would revolutionize global TB control programs. Fourth, and perhaps most speculative, an effective post-exposure vaccine that could be administered to individuals with latent TB infection to prevent subsequent reactivation. Although this approach is fraught with conceptual and practical challenges, a recent study suggests that it may be feasible. Using the murine Cornell model of chemotherapy-induced latency, Lowrie and colleagues demonstrated that post-therapy relapse could be prevented by vaccinating the treated mice with a DNA vaccine encoding the mycobacterial Hsp65 heat shock protein.102 Remarkably, severe immune suppression failed to reactivate the vaccinated animals, suggesting that the immune boost provided by vaccination allowed the complete eradication of bacteria from the tissues. Although it must be cautioned that DNA vaccination in humans has so far yielded rather disappointing results,103 and the safety and efficacy of post exposure vaccination have been challenged by others,107 an important ‘‘proof of principle’’ has been established: that post-exposure vaccination, alone or in combination with drug therapyFcould provide a more rapid and effective intervention against latent TB. Development of new interventions against latent TB infection will hinge on an improved understanding of the mechanisms that allow M. tuberculosis to persist in the face of host immunity or chemotherapy. It is not yet clear whether these forms of persistence are associated with the same or distinct cellular processes. Recent important advances have been made in identifying M. tuberculosis ‘‘persistence factors’’ that promote long-term survival in the mouse model of TB. However, it is not yet clear whether targeting these persistence factors with new drugs will actually promote more rapid killing of bacteria in vivo; nor is it clear whether persistence factors that are essential in the mouse model will likewise be important in the context of human infection. The Cornell model has been a useful tool for the study of chemotherapy-induced latency in the mouse, but this form of artificially induced latency may not be relevant to latency in humans, which is typically brought about by the unaided immune response.82 A major challenge for future research will be to take findings from model systems and prove their relevance in humans. With recent spectacular advances in technology opening, for the first time, the possibility of exploring the biology of M. tuberculosis in its natural habitat, the human lung, this promises to become an area of vigorous research activity in the near future.

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23. Medlar EM, Bernstein S, Steward DM. A bacteriologic study of resected tuberculous lesions. Am Rev Tuberc 1952;66:36–43. 24. American Trudeau Society. Report of a panel discussion on survival, revival of tubercle bacilli in healed and tuberculous lesions. Am Rev Tuberc 1953; 68: 477–95. 25. Hobby GL, Auerbach O, Lenert TF, et al. The late emergence of M. tuberculosis in liquid cultures of pulmonary lesions resected from humans. Am Rev Tuberc 1954;70:191–218. 26. Loring WE, Vandiviere HM. The treated pulmonary lesion and its tubercle bacillus. I. Pathology and pathogenesis. Am J Med Sci 1956;232:20–9. 27. Vandiviere HM, Loring WE, Melvin I, et al. The treated pulmonary lesion and its tubercle bacillus. II. The death and resurrection. Am J Med Sci 1956;232:30–7. 28. Wayne LG. The bacteriology of resected tuberculous pulmonary lesions. I. The effect of interval between reversal of infectiousness and subsequent surgery. Am Rev Tuberc Pulm Dis 1956;74:376–87. 29. McCune RM, Tompsett R. Fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. II. The conversion of tuberculosis infection to the latent state by the administration of tuberculosis and a companion drug. J Exp Med 1956; 104:763–802. 30. McCune RM, Tompsett R, McDermott W. Fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. I. The persistence of drug susceptible bacilli in the tissues despite prolonged antimicrobial therapy. J Exp Med 1956;104: 737–62. 31. McCune RM, Feldman FM, Lambert HP, et al. Microbial persistence II. Characteristics of the sterile state of tubercle bacilli. J Exp Med 1966;123:469–86. 32. McCune RM, Feldman FM, Lambert HP, et al. Microbial persistence I. The capacity of tubercle bacilli to survive sterilization in mouse tissues. J Exp Med 1966;123:445–68. 33. Barclay WR, Ebert RH, Le Roy GV, et al. Distribution and excretion of radioactive isoniazid in tuberculosis patients. JAMA 1953;151:1384–8. 34. Haapanen JH, Kass I, Gensini G, et al. Studies on the gaseous content of tuberculous cavities. Am Rev Respir Dis 1959;80:1–5. 35. Hobby GL, Lenert TF. The in vitro action of antituberculous agents against multiplying and non-multiplying microbial cells. Am Rev Tuberc 1957;76:1031–48. 36. Hobby GL, Meyer K, Chaffee E. Observations on the mechanism of action of penicillin. Proc Soc Exp Biol Med 1942;50:281–5. 37. Tomasz A, Albino A, Zanati E. Multiple antibiotic resistance in a bacterium with suppressed autolytic system. Nature 1970;227:138–40. 38. Tuomanen E. Phenotypic tolerance: the search for betalactam antibiotics that kill nongrowing bacteria. Rev Infect Dis 1986;8:S279–91. 39. Wallis RS, Patil S, Cheon SH, et al. Drug tolerance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 1999;43:2600–6. 40. Ho. ner zu Bentrup K, Russell DG. Mycobacterial persistence: adaptation to a changing environment. Trends Microbiol 2001;9:597–605. 41. Dahl JL, Kraus CN, Boshoff HI, et al. The role of Rel Mtbmediated adaption to stationary phase in long-term persistence of Mycobacterium tuberculosis in mice. Proc Natl Acad Sci USA 2003;100:10026–31.

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