Nonreplicating persistence of mycobacterium tuberculosis.

There is ample clinical evidence, as well as evidence from animal experiments, that Mycobacterium tuberculosis can persist in tissues for months to decades without replicating, yet with the ability to resume growth and activate disease. Our knowledge of both macrophage physiology and the nature of tuberculous lesions in man and animals suggests that hypoxia is a major factor in inducing nonreplicating persistence (NRP) of tubercle bacilli. In vitro models reinforce this conclusion and provide insights into mechanisms that make NRP possible. There is evidence from in vitro models that the strategies employed by the bacilli to permit hypoxic NRP include restriction of biosynthetic activity to conserve energy, induction of alternative energy pathways, and stabilization of essential cell components to lessen the need for repair or replacement.

Microaerophilic induction of the alpha-crystallin chaperone protein homologue (hspX) mRNA of Mycobacterium tuberculosis.

Among the products that are expressed when Mycobacterium tuberculosis undergoes hypoxic shiftdown to nonreplicating persistence (NRP) is the alpha-crystallin chaperone protein homologue (Acr). This expression coincides with the previously reported appearance of a respiratory type of nitrate reductase activity, the increase in glycine dehydrogenase activity, and the production of a unique antigen, URB-1. In a timed sampling study, using a slowly stirred oxygen depletion culture model, we have demonstrated that the hspX mRNA that codes for Acr protein as well as the protein itself is induced just as the bacilli enter the microaerophilic NRP stage 1 (NRP-1). In contrast to the induction observed for hspX mRNA, levels of 16S rRNA, fbpB mRNA (encoding the 85B alpha antigen), and aroB mRNA (encoding dehydroquinate synthase) demonstrate relatively small to no change upon entering NRP-1. Acr protein was shown to be identical to URB-1 by Western analysis with anti-URB-1 antibody. The fact that antibody to Acr is found in a high percentage of tuberculosis patients suggests that the hypoxic shiftdown of tubercle bacilli to the NRP state that occurs in vitro, resulting in production of the alpha-crystallin protein, occurs in vivo as well. Simultaneous abrupt increases in hspX mRNA and Acr protein suggest that Acr protein expression is controlled at the level of transcription.

In Vitro Model of Hypoxically Induced Nonreplicating Persistence of Mycobacterium tuberculosis.

Great progress has been made in the latter half of the twentieth century in the understanding of the immunology of tuberculosis and of strategies for chemotherapeutic management of this disease. Indeed, given the evidence that the dominant, and perhaps sole, ecologic niche of Mycobacterium tuberculosis is the infected human host, it seemed reasonable to hope that the disease could not only be controlled, but eradicated by the end of this century (1). These hopes are dashed by the periodic resurgence of tuberculosis in various populations. Undoubtedly socioeconomic factors have played a major role in the failure to eradicate this disease, but another, neglected, factor is the apparent ability of the tubercle bacillus to remain in a relatively quiescent state in the host, neither replicating and causing progression of disease, nor dying off and disappearing from the host's tissues, in spite of apparently adequate immune responses and aggressive chemotherapy.

Nitrate reduction as a marker for hypoxic shiftdown of Mycobacterium tuberculosis.

SETTING: In vitro cultures. OBJECTIVE: To characterize nitrate reduction during aerobic growth and hypoxic shiftdown to non-replicating persistence of Mycobacterium tuberculosis cultures. DESIGN: The rates of reduction of nitrate to nitrite were measured in cultures of M. tuberculosis growing aerobically or undergoing hypoxic shiftdown. RESULTS: Tubercle bacilli growing aerobically in the presence of nitrate reduce nitrate at a rate proportional to the substrate concentration, continuing until the substrate is exhausted. When the bacilli in an oxygen restricted model enter microaerophilic non-replicating persistence (NRP) stage 1, they exhibit a marked increase in rate of nitrate reduction that is independent of substrate concentration, and terminates by feedback inhibition when the concentration of nitrite produced approaches 2.5 mM. When bacilli in the oxygen restricted model are not supplemented with nitrate until they enter microaerophilic NRP stage 1, they exhibit an induction period before the rapid nitrate reduction starts. When the nitrate is not added until the bacilli have entered the anaerobic NRP stage 2, reduction of the substrate starts immediately. Nitrite is not reduced by M. tuberculosis in any stage of its growth or NRP. CONCLUSION: The hypoxically induced nitrate reduction probably serves a respiratory function in supporting hypoxic shiftdown of M. tuberculosis from aerobic growth to non-replication persistence and represents a useful new marker for monitoring that shiftdown. This response may help the bacilli survive in oxygen depleted regions of inflammatory or necrotic tissue, where nitrate can occur as a degradation product of nitric oxide.

An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence.

It was demonstrated previously that abrupt transfer of vigorously aerated cultures of Mycobacterium tuberculosis to anaerobic conditions resulted in their rapid death, but gradual depletion of available O2 permitted expression of increased tolerance to anaerobiosis. Those studies used a model based on adaptation of unagitated bacilli as they settled through a self-generated O2 gradient, but the model did not permit examination of homogeneous populations of bacilli during discrete stages in that adaptation. The present report describes a model based on culture of tubercle bacilli in deep liquid medium with very gentle stirring that keeps them in uniform dispersion while controlling the rate at which O2 is depleted. In this model, at least two stages of nonreplicating persistence were seen. The shift into first stage, designated NRP stage 1, occurred abruptly at a point when the declining dissolved O2 level approached 1% saturation. This microaerophilic stage was characterized by a slow rate of increase in turbidity without a corresponding increase in numbers of CFU or synthesis of DNA. However, a high rate of production of glycine dehydrogenase was initiated and sustained while the bacilli were in this state, and a steady ATP concentration was maintained. When the dissolved O2 content of the culture dropped below about 0.06% saturation, the bacilli shifted down abruptly to an anaerobic stage, designated NRP stage 2, in which no further increase in turbidity was seen and the concentration of glycine dehydrogenase declined markedly. The ability of bacilli in NRP stage 2 to survive anaerobically was dependent in part on having spent sufficient transit time in NRP stage 1. The effects of four antimicrobial agents on the bacilli depended on which of the different physiologic stages the bacilli occupied at a given time and reflected the recognized modes of action of these agents. It is suggested that the ability to shift down into one or both of the two nonreplicating stages, corresponding to microaerophilic and anaerobic persistence, is responsible for the ability of tubercle bacilli to lie dormant in the host for long periods of time, with the capacity to revive and activate disease at a later time. The model described here holds promise as a tool to help clarify events at the molecular level that permit the bacilli to persist under adverse conditions and to resume growth when conditions become favorable. The culture model presented here is also useful for screening drugs for the ability to kill tubercle bacilli in their different stages of nonreplicating persistence.

Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of the International Working Group on Mycobacterial Taxonomy.

During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 51 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16S rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjectum, a pathogen that resembles the nonpathogen Mycobacterium gordonae phenotypically. We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.

Dormancy of Mycobacterium tuberculosis and latency of disease.

There is ample circumstantial evidence from observation of the natural history of tuberculosis in humans and experimental animals that Mycobacterium tuberculosis is capable of adapting to prolonged periods of dormancy in tissues, and that these dormant bacilli are responsible for latency of the disease itself. Furthermore, the dormant bacilli are resistant to killing by antimycobacterial agents. A systematic evaluation of the mechanism of dormancy, and of attempts to abrogate latency will require a better understanding of the physiologic events that attend the shiftdown into dormancy. There are probably two or more stages in the shiftdown of Mycobacterium tuberculosis from active replication to dormancy as bacilli in unagitated cultures settle through a self-generated O2 gradient into a sediment where O2 is severely limited. One step involves a shift from rapid to slow replication. The other involves complete shutdown of replication, but not death. Presumably this last step includes completion of a round of DNA synthesis. The shiftup on resumption of aeration includes at least three discrete sequential steps, the production of RNA, the ensuing synchronized cell division and, finally, the initiation of a new round of synthesis of DNA. Three markers of the process of shiftdown of Mycobacterium tuberculosis to dormancy have been described, namely the changes in tolerance to anaerobiosis, the production of a unique antigen and the ten-fold increase in glycine dehydrogenase production. Additional markers represented in the shiftup and shiftdown process may yet be discovered, and determination of their specific functions should provide insights into the mechanisms of dormancy and latency in tuberculosis, and into strategies for preventing reactivation of the bacilli and development of disease.

Metronidazole is bactericidal to dormant cells of Mycobacterium tuberculosis.

Very abrupt exposure to anaerobic conditions has a lethal effect on actively growing cultures of Mycobacterium tuberculosis. However, incubation under conditions in which oxygen is depleted gradually causes M. tuberculosis to shift down from active replication to dormancy. The dormant bacilli are resistant to the bactericidal effects of anaerobiosis and also exhibit partial or complete resistance to the bactericidal effects of isoniazid and rifampin. On the other hand, metronidazole, a drug specific for anaerobes, kills dormant tubercle bacilli under anaerobic conditions, but it has no effect on actively growing aerobic cultures. The lethal effect of metronidazole under anaerobic conditions is enhanced by rifampin. The possible implications of these findings on the phenomenon of latency in tuberculosis are discussed.

Serovar determination and molecular taxonomic correlation in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum: a cooperative study of the International Working Group on Mycobacterial Taxonomy.

A cooperative study was conducted by the International Working Group on Mycobacterial Taxonomy to correlate the agglutination serovar designations of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum strains with the species ascriptions of these organisms according to molecular criteria and cultural properties and to assess the reproducibility of serovar determinations for a set of 63 reference strains of these species. Among the molecular criteria, the level of agreement between results obtained with nucleic acid probes and T-catalase serology results was 94% for strains of M. avium and M. intracellulare. Nucleic acid probes were not available for M. scrofulaceum, but none of the 10 strains ascribed to this species on the basis of catalase serology data reacted with a nucleic acid probe for M. avium or M. intracellulare. Ascription to a species on the basis of mycolic acid high-performance liquid chromatography patterns was in agreement with catalase serology results in 86% of the cases examined. Most strains belonging to serovars 1 through 6 and 8 through 11 were identified by molecular criteria as M. avium, most strains belonging to serovars 7, 12 through 20, 23, and 25 were identified as M. intracellulare, and most strains belonging to serovars 41 through 43 were identified as M. scrofulaceum, in agreement with common current practice. Evidence for assigning serovar 27 to M. scrofulaceum was obtained. However, two strains of a given serovar may, on occasion, be placed in different species. The dominant species assignments for strains belonging to serovars 21, 24, 26, and 28 remain unresolved.(ABSTRACT TRUNCATED AT 250 WORDS)

The impact of new technology on the laboratory's contribution to the diagnosis and management of mycobacterial disease.

From the time of the discovery of the tubercle bacillus in the late nineteenth century until the introduction of chemotherapy in the mid-twentieth, the role of the laboratory in the management of tuberculosis was limited because the treatment of the disease was nonspecific. The advent of specific chemotherapy and the recognition of human diseases caused by a number of mycobacterial species other than M. tuberculosis increased the scope and importance of the clinical laboratory in guiding the diagnosis and management of mycobacterial disease. This included the isolation of mycobacteria, the identification of the isolates, the determination of their susceptibilities to chemotherapeutic agents and their subtyping for epidemiologic purposes. In spite of the enhanced role it has played in the past forty years, the laboratory's contribution has been impeded by the slow growth of mycobacteria, which causes delays of weeks or months between submission of a specimen and the availability of a definitive report. In the meantime both the urgency and the complexity of diagnosis and management of mycobacterial disease have increased with the emergence of epidemics of these diseases associated with the acquired immunodeficiency syndrome (AIDS). This development has also increased the need for recognition of tubercle bacilli in such specimens as blood and stools, which were only infrequently studied in past years. Recent developments in microchemical and immunologic technology, and especially molecular biology, are greatly reducing the time needed to get information that contributes to diagnosis and management of mycobacterial disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoglobulin A (IgA) and IgG serum antibodies to mycobacterial antigens in Crohn's disease patients and their relatives.

Sera from patients with Crohn's disease, their relatives, their spouses, and unrelated healthy controls were assayed by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgA antibodies to Mycobacterium tuberculosis, M. avium, and M. gordonae. The patients had significantly higher IgA responses to mycobacterial antigens than did either their relatives or the controls. On the other hand, both the patients and their relatives had significantly higher IgG responses against these antigens than did the controls. The elevated IgA response was more pronounced against isopentanol-extracted whole bacterial cells than it was against soluble protein extracts, and it appeared to be directed against fixed surface antigens that lie under the loosely bound peptidoglycolipid or glycolipid antigens of mycobacteria.

Agents of newly recognized or infrequently encountered mycobacterial diseases.

This paper reviews recent information on the systematics and clinical significance of potentially pathogenic environmental mycobacteria. A short history of these mycobacteria is given. Information on species for which clinical and systematic aspects have already been well documented, i.e., Mycobacterium kansasii, M. marinum, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi, and members of the M. fortuitum complex, is updated. Although the M. avium complex was extensively reviewed in earlier literature, major new systematic and clinical information is presented in some detail. Species that have received very limited prior coverage, i.e., M. asiaticum, M. haemophilum, M. malmoense, and M. shimoidei, are the main subjects of this review and are discussed in detail. The rare infections attributed to species that are normally considered nonpathogenic, i.e., M. gastri, M. gordonae, the M. terrae complex, and most of the rapidly growing mycobacteria outside of the M. fortuitum complex, are critically reviewed. Finally, suggestions are offered for practical measures that can minimize the risk of failing to isolate or misidentifying some of the more obscure potentially pathogenic environmental mycobacteria that are only infrequently recognized.

Fourth report of the cooperative, open-ended study of slowly growing mycobacteria by the International Working Group on Mycobacterial Taxonomy.

The open-ended study of the International Working Group on Mycobacterial Taxonomy is an ongoing project to characterize slowly growing strains of mycobacteria that do not belong to well-established or thoroughly characterized species. In this fourth report we describe two numerical taxonomic clusters that represent subspecies or biovars of Mycobacterium simiae, one cluster that encompasses the erstwhile type strain of the presently invalid species "Mycobacterium paraffinicum," one cluster that is phenotypically very similar to Mycobacterium avium and Mycobacterium intracellulare but may be a separate genospecies, one cluster that appears to be phenotypically distinct from M. avium but reacts with a nucleic acid probe specific for M. avium, and three tentatively defined clusters in proximity to a cluster that encompasses the type strain of Mycobacterium malmoense. Of special practical interest is the fact that one of the latter three clusters is composed of clinically significant scotochromogenic bacteria that can be misidentified as the nonpathogenic organism Mycobacterium gordonae if insufficient biochemical tests are performed.

Antimycobacterial antibody levels in pleural fluid as reflection of passive diffusion from serum.

The objective of this study was the prospective evaluation of the relationship between serum and pleural fluid antibody levels to mycobacterial antigens and their role in the diagnosis of tuberculous pleuritis. The setting was a tertiary care medical center. Thirteen patients with tuberculous pleuritis and 53 control subjects with pleural effusion (22 with carcinoma, 17 with cardiac failure, and 14 with empyema or parapneumonic effusion) were studied. The level of IgG was measured by ELISA. The median titers of antibody to both Mycobacterium tuberculosis and M avium were significantly higher in the serum and pleural fluid of the patients with tuberculosis than in the control patients. There was a very close relationship between the levels of M tuberculosis (r = 0.95) and M avium (r = 0.94) antibodies in the serum and pleural fluid. We concluded that the levels of antimycobacterial IgG in pleural fluid, adjusted to constant protein concentration, are very closely related to the serum levels. Therefore, these antibodies in the pleural fluid probably result from passive diffusion from serum and not local production. Measurement of pleural fluid antibody levels will not add diagnostic sensitivity or specificity to that achieved with serodiagnosis.

Antibodies to mycobacterial peptidoglycolipid and to crude protein antigens in sera from different categories of human subjects.

Sera from patients with disease caused by the Mycobacterium avium complex (M. avium and M. intracellulare), M. kansasii, or M. tuberculosis and from subjects who did not have a mycobacterial disease were tested by enzyme-linked immunosorbent assay against peptidoglycolipid antigens representing each of the 15 most common serovars of the M. avium complex and against crude protein antigen extracts of M. avium and M. tuberculosis. The highly specific peptidoglycolipid antigens yielded positive reactions in 83% of M. avium complex patients, 57% of active-tuberculosis patients, and 14% of subjects without mycobacterial disease. Reactions to more than 1 of the 15 peptidoglycolipid antigens were found only in patients with infections caused by mycobacteria, suggesting that a mycobacterial pulmonary lesion is readily colonized by mycobacteria other than the one that initiated the lesion. The two crude mycobacterial protein antigens were highly cross-reactive, with little if any capacity to discriminate between infections caused by any of the mycobacteria studied. Moreover, they did not appear to be more sensitive than the peptidoglycolipids. The data suggest that it is unlikely that a practical and reliable serological test can be developed that will distinguish between transient subclinical infection and significant disease caused by common environmental mycobacteria, such as members of the M. avium complex. Success in developing such a test for nonenvironmental mycobacteria, such as M. tuberculosis, appears more likely.

Detection of a novel catalase in extracts of Mycobacterium avium and Mycobacterium intracellulare.

A novel class of catalase, which differs from the previously described M- and T-catalases of mycobacteria, was detected in strains of Mycobacterium avium and M. intracellulare. Designated A-catalase, this enzyme resisted inactivation at 68 degrees C, was inactivated by 3-amino-1,2,4-triazole (aminotriazole), and exhibited no peroxidase activity. All of these properties distinguished the enzyme from T-catalase. The A-catalase exhibited a Km of 70 mM H2O2, which is between the upper and lower extremes of the ranges reported for T- and M-catalases, respectively. The A-catalase appeared to be more hydrophobic than M-catalase and did not react with antiserum to a representative sample of this class. The banding patterns of T- and M-catalases seen by polyacrylamide gel electrophoresis (PAGE) were essentially unaffected by the incorporation of sodium dodecyl sulfate (SDS) into the PAGE system, whereas the single band of A-catalase seen by PAGE without SDS resolved into as many as five bands in the presence of SDS; these bands were all of slower mobility than the original band. The banding pattern seen with SDS appeared to be related more to counterion charge effects than to molecular size increases that could be attributed to SDS complexed to the protein. It remains to be determined whether the multiple A-catalase bands reflect different proteins or different SDS micellar complexes of a single protein.

Intrinsic catalase dot blot immunoassay for identification of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare.

The heat-labile T class of mycobacterial catalase exhibits peroxidase activity with some substrates. Most species of mycobacteria produce T-catalase, which is serologically characterized by a combination of shared epitopes and unique, species-specific epitopes. Antibodies to T-catalases from Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare have been cross absorbed with T-catalases from heterologous species and applied as dots to nitrocellulose membranes. When these membranes were incubated with crude sonic extracts of 93 strains of mycobacteria that produce sufficient T-catalase, and were then exposed to 3,3'-diaminobenzidine peroxidase substrate, only those extracts made from one of the three species represented yielded a discrete brown dot at the site of the corresponding globulin. The sensitivity of the test was at least 96.5%, and the specificity was in excess of 99.5%.