Transport Assays and Permeability in Pathogenic Mycobacteria

Mycobacteria produce an effective permeability layer that consists of a mycolic acid–containing cell wall. This protection confers a natural resistance to many chemical agents and results in a low permeability toward both hydrophilic and lipophilic agents. The permeability of cells is classically measured using methods that generally need cell suspensions and are hazardous with pathogens (e.g., nutrient and antibiotic uptake). A major problem encountered with mycobacteria is their propensity to form aggregates; the addition of detergent to the cell suspension is not recommended as this disorganizes the cell envelope, rendering it more permeable to antibiotics. To circumvent this problem, growing cells are uniformly labeled with [³ H]-uracil, allowing a quantification of the aliquots; then, the uptake of [¹⁴ C]-labeled probes is followed during the first minutes. To avoid the generation of aerosols associated with the commonly used filtration methods, centrifugation through an oil mixture is the preferred alternative technique for use with Mycobacterium tuberculosis .