Higher-order organization of bacterial cords and an amyloid-like matrix define the Mycobacterium tuberculosis biofilm

Bacterial biofilms generate collective properties that cannot be explained by individual cells alone. Yet the structural principles that organize these communities and their relationship to drug tolerance remain poorly understood, including in major human pathogens such as Mycobacterium tuberculosis (Mtb). Here we show that submerged Mtb biofilms form organized multicellular architectures in which bacterial cords are integrated with a composite extracellular matrix. The virulence lipid phthiocerol dimycocerosate (PDIM) governs the higher-order organization of cords, proteinaceous matrix interactions support cohesion and attachment, an amyloid-like component contributes to biofilm integrity and establishment, and the ESX-1 secretion system increases matrix biochemical complexity. Biofilm growth conferred antibiotic tolerance across genetic backgrounds, while PDIM-dependent organization provided additional protection against first-line anti-tuberculosis drugs, most clearly isoniazid. Targeting amyloid assembly with epigallocatechin gallate impaired biofilm establishment without inhibiting planktonic growth. Together, these findings establish a cording-centred framework linking bacterial surface composition, cellular organization and extracellular matrix assembly to antibiotic tolerance, and show how resolving higher-order biofilm architecture can reveal processes amenable to chemical perturbation.