Contingencies in biofilm adaptation of Mycobacterium tuberculosis

Biofilms are structured microbial communities that offer protection from a range of environmental stressors. Studies of experimental evolution within biofilms have yielded important insights into mechanisms of biofilm formation, as well as fundamental principles governing bacterial adaptation within these structured communities. Building on research using tractable species, our lab created a model for studying biofilm adaptation in Mycobacterium tuberculosis (M. tb), a fastidious, slow growing and lethal pathogen of humans. Here, we evolved eighteen M. tb populations arising from six parental genetic backgrounds under biofilm selection to investigate diversity in mechanisms of M. tb biofilm adaptation. We found that fine-scale differences among strains at the initiation of the experiment influenced the degree of replicability in their evolution. Adaptive paths were highly parallel for some strains, whereas others evolved distinct mutations across iterations of the experiment. Our data suggest that differences in replicability arise from mutational biases and variable fitness impacts of mutations across genetic backgrounds. Comparison of our results with genomic data from M. tb populations within hosts with tuberculosis (TB) revealed that several mutations associated with biofilm selection are also among the most common to emerge during natural infection. These biofilm-associated variants are not maintained in natural M. tb populations, suggesting that biofilm selection in our model mimics selection pressures that are transiently encountered by M. tb during specific phases of infection. Overall, these results support development of biofilm directed therapies for TB and demonstrate the importance of subtle genetic variation in shaping M. tb responses to changing selection pressures. SignificanceMycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), a difficult-to-treat, persistent infection with high mortality. One cause of this persistence is the ability of M. tb to form biofilms, aggregated structures capable of resisting antibiotics and host defenses. Here, we used an evolutionary model to investigate mechanisms of M. tb biofilm formation. We found adaptation to biofilm growth to be affected by mutational biases and interactions among mutations. Our simple in vitro model appears to mimic aspects of natural infection, as identical mutations emerge in the laboratory under biofilm selection and within hosts with TB. These results expand our knowledge of M. tb biofilm development and inform our understanding of how this bacterium responds to novel selection pressures.