Impact of growth media on vancomycin susceptibility and mycobacterial cell wall integrity

Tuberculosis remains the leading cause of death by a single infectious agent, Mycobacterium tuberculosis , in the world. To tackle this disease, efforts are conducted, among others, to improve diagnosis coverage and treatment access, to develop an improved vaccine, and to find new antituberculous drugs or drug regimens. In vitro , bacteria usually tend to lose virulence factors to improve their fitness, mostly observed by an increased growth. This is also the case for M. tuberculosis strains and clinical isolates prone to lose phthiocerol dimycocerosates in the upper leaflet of their mycomembrane. This loss affects the cell wall integrity, as observed by an increased antimycobacterial activity of vancomycin, providing a potential bias in drug susceptibility testing. Here, we showed that M. tuberculosis culture conditions, such as a single subculture on 7H11 supplemented with oleic acid–albumin–dextrose–catalase, can restore vancomycin resistance and cell wall integrity but can also impact routine drug susceptibility assay by broth microdilution. IMPORTANCE Vancomycin susceptibility testing can identify Mycobacterium tuberculosis subcultures with compromised cell wall integrity due to phthiocerol dimycocerosates loss. A single pre-culture on potato-Sauton medium or on 7H11 supplemented with oleic acid–albumin–dextrose–catalase can restore cell wall integrity. This study underscores the importance of standardized culture protocols for reliable M. tuberculosis drug susceptibility testing.