Non-uniform growth probability of Mycobacterium tuberculosis across inoculum densities partially restored by culture filtrate: Implications for supplemented growth assays.

Differentially culturable (DC) Mycobacterium tuberculosis (Mtb) phenotypes reduce the sensitivity of sputum culture and may be associated with adverse treatment outcomes in tuberculosis patients. Detecting and quantifying DC Mtb in clinical samples remains an important research objective; current approaches rely on culture filtrate (CF) supplemented growth assays that assume CF selectively resuscitates DC Mtb rather than broadly stimulating growth from low density inocula - an untested assumption for Mtb. We performed a half-logarithmic dilution series of Mtb from approximately 10,000-0 total bacilli, culturing each inoculum in either standard media (7H9) or CF and monitoring growth by optical density. Odds of growth increased sixfold for every tenfold increase in inoculum size. CF increased the odds of growth fivefold and reduced the time-to-positivity by 344 h (∼14 days) compared to 7H9 alone. However, CF's growth-promoting effects diminished with increasing inoculum size, becoming negligible at approximately 70,000 bacilli/mL. Notably, CF broadly altered Mtb cell physiology, producing shorter bacilli that were less likely to form aggregates or incorporate the mycomembrane probe, DMN-trehalose. These data indicate that Mtb is poorly culturable from low inoculum densities under conditions tested, with no growth occurring in wells with ≤30 total bacilli, a limitation only partially overcome by CF. Moreover, while CF promotes Mtb growth, its effects on bacillary physiology suggest an impact extending beyond simply resuscitation of DC Mtb. Improved methods are needed for detecting DC Mtb phenotypes, as current CF-based assays may not distinguish selective resuscitation from broader growth-promoting effects.