Jansy Sarathy’s Hackensack Meridian Center for Discovery & Innovation lab developed a novel in vitro model that mimics the caseum environment found in tuberculosis patients, harboring drug-tolerant Mycobacterium tuberculosis. A major obstacle to effective tuberculosis chemotherapy is the presence of persistent bacterial populations residing in specific host microenvironments. Caseum, the necrotic core of tuberculosis granulomas and cavities, harbors high burdens of drug-tolerant M. tuberculosis bacilli, making them particularly difficult to sterilize. This drug tolerance necessitates prolonged treatment durations and increases the risk of treatment failure. Previous in vitro models of nonreplicating persistent M. tuberculosis often focused on single environmental stresses like oxygen starvation or nutrient deprivation, which may not fully represent the complex caseum environment. The lab protocols for generating this caseum surrogate using lipid-rich macrophages enable researchers to study the nonreplicating persistent form of the bacteria and evaluate the effectiveness of antimicrobial agents against it. This physiologically relevant model offers advantages over existing in vitro methods by more accurately replicating the drug tolerance observed in the native caseous environment, ultimately aiming to reduce reliance on in vivo studies for drug screening and target identification. The described protocols include methods for caseum surrogate preparation, M. tuberculosis inoculation, preadaptation, and various drug susceptibility testing assays, including higher-throughput screening options.
Xie M, Osiecki P, Rodriguez S, Dartois V, Sarathy J. A Physiologically Relevant In Vitro Model of Nonreplicating Persistent Mycobacterium tuberculosis in Caseum. Curr Protoc. 2025 Mar;5(3):e70118. doi: 10.1002/cpz1.70118. PMID: 40056090. https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.70118
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