SummarySuccessful treatment of human tuberculosis requires 6-9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms -organized communities of surface-attached cells -but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics.

Many of the current antimycobacterial agents require some form of cellular activation unmasking reactive groups, which in turn will bind to their specific targets. Therefore, understanding the mechanisms of activation of current antimycobacterials not only helps to decipher mechanisms of drug resistance but may also facilitate the development of alternative activation strategies or of analogues that do not require such processes. Herein, through the use of genetically defined strains of Mycobacterium bovis BCG we provide evidence that EthA, previously shown to activate ethionamide, also converts isoxyl (ISO) and thiacetazone (TAC) into reactive species. These results were further supported by the development of an in vitro assay using purified recombinant EthA, which allowed direct assessment of the metabolism of ISO. Interestingly, biochemical analysis of [ 14 C]acetate-labeled cultures suggested that all of these EthA-activated drugs inhibit mycolic acid biosynthesis via different mechanisms through binding to specific targets. This report is also the first description of the molecular mechanism of action of TAC, a thiosemicarbazone antimicrobial agent that is still used in the treatment of tuberculosis as a second-line drug in many developing countries. Altogether, the results suggest that EthA is a common activator of thiocarbamide-containing drugs. The broad specificity of EthA can now be used to improve the activation process of these drugs, which may help overcome the toxicity problems associated with clinical thiocarbamide use.Despite the availability of effective therapies, tuberculosis (TB), caused by Mycobacterium tuberculosis, is still a leading cause of death (11). The human immunodeficiency virus pandemic, which contributes substantially to the morbidity and mortality from TB, and the emergence of multidrug-resistant strains of M. tuberculosis (23) have compounded the problem. Although infections by drug-sensitive strains can be successfully cured (7), the emergence of drug resistance has prompted new drug research, particularly the search for new drug targets and the definition of mechanisms of drug resistance (16). When TB cases cannot be treated by first-line protocols due to resistance issues, the last resort for combating multidrug-resistant infections relies on the action of second-line antitubercular drugs.Work from the last decade has revealed M. tuberculosis to be unique among bacteria in that several drugs require activation in situ to become inhibitory. Drugs such as isoniazid (INH), ethionamide (ETH), and pyrazinamide (PZA) all require activation for activity against M. tuberculosis, and resistance can be mediated by mutations that eliminate the activation step. Such inactivation has been demonstrated for the catalase-peroxidase KatG in INH resistance (33), the nicotinamidase-peroxidase PncA in PZA resistance (24), and the flavin adenine dinucleotide (FAD)-containing Baeyer-Villiger monooxygenase EthA in ETH resistance (3, 9, 30). Interestingly, both activated forms of INH and ETH targe...