Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.Tuberculosis is an important infectious disease, with over 1 billion people subclinically infected and the 3 million new cases each year resulting in 7% of the total number of deaths (20). Case finding and treatment are the main control measures, but chemotherapy takes 6 months to achieve a cure because bacteria persist during chemotherapy and cause relapse (2 to 5% in 5 years) after the end of treatment (14). These persistent bacteria are usually drug sensitive at relapse, and so their resistance to chemotherapy is phenotypic (17) (or tolerant) rather than genetic. It is suggested that an altered physiological state of persistent Mycobacterium tuberculosis accounts for its tolerance to drugs as well as the ability to survive in the host for many years. Persistence is likely to be a combined effect of both the immune system and bacterial physiology, resulting in what is generally referred to as a latent state (1). A key question is whether the metabolism of persistent bacteria is switched off with no cell division (spore-like) or, alternatively, is active (30).The first major advance in the detection of quiescent M. tuberculosis was the development of the Cornell mouse model (25). In this model, animals are infected with M. tuberculosis and treated for 3 months with chemotherapy which renders the tissues sterile; then steroids are administered, which leads to relapse in a high proportion of the mice. Immediately after the end of antibiotic therapy, the persistent bacteria are invisible by microscopy and are uncultivable, but about 10 5 genome equivalents of M. tuberculosis DNA per ml of tissue in the organs have been detected by DNA amplification (4). The presence of DNA could be indicative of dead bacilli or, possibly, of live bacilli which can multiply when the host immune system becomes weakened by steroids. Since DNA can be detected in dead bacteria (9,16,24), targeting genomic DNA is not suitable for the ...

cThe treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time frame, with one case sequenced retrospectively. WGS identified mutations in the M. tuberculosis genes associated with antibiotic resistance that are likely to be responsible for the phenotypic resistance. Thus, an evidence base was developed to inform the clinical decisions made around antibiotic treatment over prolonged periods. All strains in this study belonged to the East Asian (Beijing) lineage, and the strain relatedness was consistent with the expectations from the case histories, confirming one contact transmission event. We demonstrate that WGS data can be produced in a clinically relevant time scale some weeks before drug sensitivity testing (DST) data are available, and they actively help clinical decision-making through the assessment of whether an isolate (i) has a particular resistance mutation where there are absent or contradictory DST results, (ii) has no further resistance markers and therefore is unlikely to be XDR, or (iii) is identical to an isolate of known resistance (i.e., a likely transmission event). A small number of discrepancies between the genotypic predictions and phenotypic DST results are discussed in the wider context of the interpretation and reporting of WGS results. In 2013, of the 6.1 million new tuberculosis (TB) cases reported worldwide, 480,000 were defined as multidrug resistant (MDR) (1). MDR strains of Mycobacterium tuberculosis are defined as those that are resistant to the key first-line drugs, rifampin (RIF) and isoniazid (INH). Such cases need to be treated using a more complex and flexible drug selection algorithm than the standard, with a combination of at least four drugs from an available panel classified according to their mode of action, efficacy, and clinical experience, and should be further modified if other resistances become apparent (2).Extensively drug-resistant (XDR) strains of M. tuberculosis are defined as MDR strains that in addition to being RIF and INH resistant are also resistant to the two most effective remaining bactericidal drug classes, at least one fluoroquinolone (FLQ), and one second-line injectable agent (amikacin [AMI] or kanamycin [KAN], or capreomycin [CAP]). The XDR designation reflects the concern that these are even more difficult to treat. Globally, in 2014, 9.0% of MDR cases were reported as being XDR (1); these figures are likely to be underestimates, because testing and reporting practices are not universal.The overall rates of TB in the United Kingdom have been stable, at ϳ14 per 100,000 since 2005, with London having the highest regional ...

About 90% of the bacilli in sputum are persisters that can grow in liquid media but not on solid plates.

Background: Old, stationary cultures of Mycobacterium tuberculosis contain a majority of bacteria that can grow in broth cultures but cannot grow on solid medium plates. These may be in a nonreplicating, dormant growth phase. We hypothesised that a similar population might be present in chronic, murine tuberculosis.