Adaptation of <i>Mycobacterium tuberculosis</i> to biofilm growth is genetically linked to drug tolerance

ABSTRACT Mycobacterium tuberculosis (Mtb) 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 Mtb biofilms is unknown. Using a Tn-seq approach, we show here that multiple genes that are necessary for fitness of Mtb cells within biofilms, but not in planktonic cultures, are also important for their tolerance to a diverse set of stressors and antibiotics. Thus, development of Mtb biofilms appears to be associated with population enrichment, in which endogenous stresses presumably generated by challenging growth conditions within biofilm architecture select for cells that maintain tolerance to exogenous stresses including antibiotic exposure. We further observed that the intrinsic drug tolerance of constituent cells of biofilms determines the frequency of persisters: morphologically indistinguishable monoculture biofilms of a Δ pstC2A1 mutant hypersensitive to rifampicin harbor ∼20-fold fewer persisters than wild-type. These findings together allow us to propose that the selection of elite cells during biofilm development significantly contributes to the persister frequency. 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 lipopeptides (INLP). Mutation analysis indicates that INLP is necessary for the architecture development of Mtb biofilms. In summary, the study offers an insight into persistence of Mtb biofilms under antibiotic exposure, while identifying INLP as a biomarker for further investigation of this phenomenon. SIGNIFICANCE The tuberculosis (TB) pathogen Mycobacterium tuberculosis (Mtb) is one of the deadliest bacterial pathogens known to mankind, and TB treatment is inefficient. A lengthy chemotherapy for TB is attributed to a small subpopulation of Mtb bacilli exhibiting phenotypic tolerance to antibiotics. Drugs targeting these persisters are expected to shorten TB chemotherapy, but their development is dependent on in vitro growth models that reproducibly generate high frequency of persisters. Biofilms of Mtb are a suitable model for understanding the origin of persisters. Here, we provide an explanation for the elevated persister frequency in Mtb biofilms. We also identify isonitrile lipopetides as a biomarker of Mtb biofilms. These findings will facilitate further advancements of our efforts to identify and target Mtb persisters.