Keto-Mycolic Acid-Dependent Pellicle Formation Confers Tolerance to

D.S. and D.N.D. contributed equally to this article. ABSTRACT The chronic nature of tuberculosis (TB), its requirement of long duration of treatment, its ability to evade immune intervention, and its propensity to relapse after drug treatment is discontinued are reminiscent of other chronic, biofilm-associated bacterial diseases. Historically,Mycobacterium tuberculosiswas grown as a pellicle, a biofilm-like structure, at the liquid-air interface in a variety of synthetic media. Notably, the most widely administered human vaccine, BCG, is grown as a pellicle for vaccine production. However, the molecular requirements for this growth remain ill defined. Here, we demonstrate that keto-mycolic acids (keto-MA) are essential for pellicle growth, andmutants lacking in or depleted of this MA species are unable to form a pellicle. We investigated the role of the pellicle biofilm in the reduction of antibiotic sensitivity known as drug tolerance using the pellicle-defectivemmaA4mutant strain. We discovered that themmaA4mutant, which is both pellicl