Abstract
Mycobacterium tuberculosis spontaneously grows at the air-medium interface, forming pellicle biofilms, which harbor more drug-tolerant persisters than planktonic cultures. The underlying basis for increased persisters in M. tuberculosis biofilms is unknown. Using a transposon sequencing (Tn-seq) approach, we show here that multiple genes that are necessary for fitness of M. tuberculosis cells within biofilms, but not in planktonic cultures, are also implicated in tolerance of bacilli to a diverse set of stressors and antibiotics. Thus, development of M. tuberculosis biofilms appears to be associated with an enrichment of population, in which challenging growth conditions within biofilm architecture select for cells that maintain intrinsic tolerance to exogenous stresses, including antibiotic exposure. We further observed that the intrinsic drug tolerance of constituent cells of a biofilm determines the frequency of persisters. These findings together allow us to propose that the selection of elite cells during biofilm development promotes the frequency of persisters. Furthermore, probing the possibility that the population enrichment is an outcome of unique environment within biofilms, we demonstrate biofilm-specific induction in the synthesis of isonitrile lipopeptide (INLP). Mutation analysis indicates that INLP is necessary for the architecture development of M. tuberculosis biofilms. In summary, this study offers an insight into persistence of M. tuberculosis biofilms under antibiotic exposure, while identifying INLP as a potential biomarker for further investigation of this phenomenon.