Summary: Successful 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.

Significance: Mycobacterium abscessus is the most frequently isolated rapidly growing mycobacterium in human disease and recently has emerged as responsible for severe pulmonary infections in cystic fibrosis patients. However, little is known about the virulence mechanisms of this human pathogen. We adapted the zebrafish embryo as a tractable infection model to study, at a spatiotemporal level, the physiopathology of M. abscessus infection. We describe the high propensity of virulent rough variant M. abscessus to produce serpentine cords in vivo , which are not observed with the less virulent smooth variant. We demonstrate that extracellular cording allows the bacterium to withstand phagocytosis, leading to uncontrolled growth and establishment of an acute and lethal infection, thus constituting a determinant of virulence.