Oxidative damage and delayed replication allow viable Mycobacterium tuberculosis to go undetected [TnSeq]

“Viable but non-culturable” (VBNC) states pose challenges for environmental and clinical microbiology, but their biological mechanisms remain obscure. Mycobacterium tuberculosis (Mtb), the leading cause of death from infection until COVID-19, affords a striking example. Mtb can enter into a “differentially detectable” (DD) state associated with phenotypic antimicrobial resistance in which Mtb cells are viable but undetectable as colony-forming units. We found that Mtb cells enter the DD state when they undergo sublethal oxidative stress that damages their DNA, proteins, and lipids, and in addition, their replication is delayed, allowing repair. Mycobacterium bovis and BCG fail to enter the DD state under similar conditions. These findings have implications for TB latency, detection, relapse, treatment monitoring, and development of regimens that overcome phenotypic antimicrobial resistance. H37Rv Transposon-library Sequencing of outgrowth cultures after exposure to the following conditions: growth in nutrient rich media; two weeks of nutrient starvation in PBS with tyloxapol; two weeks of nutrient starvation in PBS with tyloxapol followed by 5 days of 1% DMSO exposure; two weeks of nutrient starvation in PBS with tyloxapol followed by 5 days of exposure to 100 uM rifampin. For each listed condition, the subsequent outgrowth of the transposon library was carried out on nutrient rich solid agarose media, 5 mL of nutrient rich liquid media, or 50 mL of nutrient rich liquid media. 2 experiments with 1 sample for each condition/outgrowth in each experiment.