Biofilm Associated Persistence and Drug Tolerance in Mycobacteria Within Host Microenvironments

Biofilms formed by mycobacteria, particularly Mycobacterium tuberculosis (Mtb), represent a major challenge in tuberculosis (TB) treatment due to their highly organized structure and their capacity to induce phenotypic drug tolerance. These three-dimensional bacterial aggregates are embedded in a self-produced extracellular matrix that restricts antibiotic penetration and shields bacilli from host immune responses. The resulting spatial and physiological heterogeneity within biofilms generates microenvironments that favor slow-growing or non-replicating cells, markedly reducing the efficacy of conventional antimicrobial therapies. Increasing experimental and clinical evidence supports the presence of biofilm-like mycobacterial communities in TB lesions, linking this growth mode to disease chronicity, treatment failure, and relapse. This review aims to provide an integrated overview of the biological and physiological states adopted by mycobacteria within biofilm microenvironments, with particular emphasis on the mechanisms underlying biofilm-associated drug tolerance. In addition, it critically discusses therapeutic strategies designed to overcome this tolerance, focusing on synergistic antibiotic combinations and peptide-antibiotic therapies that directly disrupt biofilm architecture, enhance drug penetration, or sensitize biofilm-embedded bacilli to antimicrobial killing.