Culture filtrate selectively promotes Mycobacterium tuberculosis growth from extremely low density inocula: implications for quantifying differentially culturable phenotypes

Differentially culturable (DC) Mycobacterium tuberculosis (Mtb) phenotypes reduce the sensitivity of sputum culture and may be associated with adverse treatment outcomes among tuberculosis patients. Accurately quantifying DC Mtb remains an important research objective, with current approaches tending to combine Most Probable Number (MPN) assays and culture filtrate (CF) supplementation. These assume that growth is equiprobable across all bacterial inoculum densities - an untested assumption for Mtb. We performed a half-logarithmic dilution series of Mtb from 70,000-0 CFU/mL, culturing each inoculum in either standard 7H9 or CF and monitoring growth by optical density. Inocula of [≥]2,000 CFU/mL were 33 times more likely to grow than inocula of [≤]700 CFU/mL. CF increased the odds of growth five-fold, and reduced the time-to-positivity by 294 hours ([~]12 days) compared to 7H9 alone. However, CFs growth-promoting effects diminished with increasing inoculum density, becoming negligible at 70,000 CFU/mL. Notably, CF broadly altered Mtb cell physiology, producing shorter bacilli that were less likely to incorporate the mycomembrane probe, DMN-trehalose. These data indicate that Mtb is poorly culturable from low inoculum densities - a limitation only partially overcome by CF. This non-uniform growth probability suggests that unsupplemented MPN assays may systematically underestimate Mtb CFU. Moreover, while CF promotes Mtb growth, its density-dependent activity and broader effects on cell physiology suggest that its influence extends beyond simply resuscitating DC Mtb. Improved methods are needed for detecting DC Mtb phenotypes, as these may support clinical care and shed light on factors that govern mycobacterial replication at different population densities. HighlightsO_LIMycobacterium tuberculosis (Mtb) culturability decreases at low inoculum densities C_LIO_LIMost Probable Number (MPN) assays may systematically underestimate Mtb CFUs C_LIO_LICulture filtrate (CF) selectively promotes Mtb growth from low densities C_LIO_LIMtb requires secreted factors in culture filtrate to initiate growth at low cell numbers C_LIO_LICF-supplemented MPN assays may be inadequate for detecting DC Mtb phenotypes C_LI