Tuberculosis (TB), a disease that is mostly caused by Mycobacterium tuberculosis, was the leading cause of death from a single infectious agent before Sars-CoV2 swept the globe. In 2020, 9.9 million people fell ill with TB and 1.5 million died from it. Treatment for drug-susceptible TB is largely effective but 6.8% of patients receiving standard treatment develop recurrent TB and treatment options are limited for rifampicin resistant and multidrug-resistant TB. Antimicrobial resistance (AMR) not only confounds treatment for TB but also for a growing number of other infections, making it a threat to human health, security and economic advancement.
AMR refers to the ability of bacteria to avoid or delay being killed by an antibiotic and can manifest into three forms: resistance, tolerance and persistence. In resistance, bacteria that acquired a stable and heritable mutation, or new genetic material, grow in the presence of an antibiotic, resulting in a shift in the minimum inhibitory concentration (MIC) that inhibits bacterial growth. Tolerance and persistence describe phenomena where bacteria survive but don’t grow in the presence of an antibiotic. In tolerance, the entire bacterial population has a slower rate of killing by an antibiotic, and it can be genetic or phenotypic in nature. In persistence, only a subpopulation of bacteria—so-called persisters—have a slower rate of killing upon exposure to lethal concentration of an antibiotic. Persisters form stochastically—at rare frequencies—in unstressed bacterial population. The frequency increases in bacterial population exposed to stresses imposed by antibiotics themselves or by host immunity.
Tolerance and persistence have major clinical consequences, including long treatment times for TB, recurrence of disease after conclusion of antibiotic therapy for many infections, and emergence of resistance. Importantly, standard clinical antibiotic susceptibility assays do not assess tolerance or persistence, and development of tools to detect these phenomena in TB patients and new drugs to target bacteria displaying them is hindered by lack of understanding of the molecular underpinnings of tolerance and persistence in Mtb. To fill this gap, we aimed to exploit the fact that some mutations (high survival mutations) lead to genetic tolerance or high persistence. The latter can affect the frequency at which persisters arise—still stochastically—in bacterial populations. To isolate high survival mutants, we developed a method that allows for their isolation and identification in vitro and in more complex biological settings. This method, called ReMIND (Recombination mediated isolation of non-dividers), allows for discrimination of resisters from non-growing survivors based on the expression of two selection markers. Using it, we are now equipped with a powerful tool to inform the molecular mechanisms by which persistent and tolerant bacteria form during infection.
