Growth arrest of <i>Mycobacterium tuberculosis</i> in acidic environments enhances their survival of antibiotic treatment

by Eun Seon Chung, William C. Johnson, Maliwan Kamkaew, Timothy A. Fitzgerald, Morgan E. McNellis, Trever C. Smith II, Srinivasan Vijay, Nguyen Thuy Thuong Thuong, Shumin Tan, Bree Beardsley Aldridge

The ability of Mycobacterium tuberculosis (Mtb) to dynamically adjust its growth behavior in response to host environments is critical for survival under immune and drug stress, but how these behaviors shift at the single-cell level remains poorly understood. Here, using high-resolution single-cell analysis, we show that Mtb adapts to acidic conditions by increasing the proportion of bacteria in a growth-arrested state, rather than uniformly slowing the growth rate of the entire population. This nongrowing subpopulation exhibits enhanced tolerance to ethambutol, highlighting its role in drug survival. Clinical strains displayed higher proportions of growth-arrested cells under both neutral and acidic conditions, suggesting that growth arrest may serve as one of the strategies for persistence during infection. While the PhoPR two-component system partially regulates this state, our RNA sequencing analysis revealed additional transcriptional regulators that are upregulated following acidic adaptation and may contribute to entry into the growth-arrested state and increased tolerance to ethambutol. Our study demonstrates that increasing the proportion of nongrowing subpopulations is an active adaptive strategy that can influence antibiotic susceptibility under acidic conditions, offering new perspectives for targeting bacterial heterogeneity in tuberculosis therapy.