Murine tuberculosis drug efficacy studies have historically monitored bacterial burden based on CFU of Mycobacterium tuberculosis in lung homogenate. In an alternative approach, a recently described molecular pharmacodynamic marker called the RS ratio quantifies drug effect on a fundamental cellular process, ongoing rRNA synthesis. Here, we evaluated the ability of different pharmacodynamic markers to distinguish between treatments in three BALB/c mouse experiments at two institutions. We confirmed that different pharmacodynamic markers measure distinct biological responses. We found that a combination of pharmacodynamic markers distinguishes between treatments better than any single marker. The combination of the RS ratio with CFU showed the greatest ability to recapitulate the rank order of regimen treatment-shortening activity, providing proof of concept that simultaneous assessment of pharmacodynamic markers measuring different properties will enhance insight gained from animal models and accelerate development of new combination regimens. These results suggest potential for a new era in which antimicrobial therapies are evaluated not only on culture-based measures of bacterial burden but also on molecular assays that indicate how drugs impact the physiological state of the pathogen.

There is a critical need for improved pharmacodynamic markers for use in human tuberculosis (TB) drug trials. Pharmacodynamic monitoring in TB has conventionally used culture or molecular methods to enumerate the burden of Mycobacterium tuberculosis organisms in sputum. A recently proposed assay called the rRNA synthesis (RS) ratio measures a fundamentally novel property, how drugs impact ongoing bacterial rRNA synthesis. Here, we evaluated RS ratio as a potential pharmacodynamic monitoring tool by testing pretreatment sputa from 38 Ugandan adults with drug-susceptible pulmonary TB. We quantified the RS ratio in paired pretreatment sputa and evaluated the relationship between the RS ratio and microbiologic and molecular markers of M. tuberculosis burden. We found that the RS ratio was highly repeatable and reproducible in sputum samples. The RS ratio was independent of M. tuberculosis burden, confirming that it measures a distinct new property. In contrast, markers of M. tuberculosis burden were strongly associated with each other. These results indicate that the RS ratio is repeatable and reproducible and provides a distinct type of information from markers of M. tuberculosis burden. IMPORTANCE This study takes a major next step toward practical application of a novel pharmacodynamic marker that we believe will have transformative implications for tuberculosis. This article follows our recent report in Nature Communications that an assay called the rRNA synthesis (RS) ratio indicates the treatment-shortening of drugs and regimens. Distinct from traditional measures of bacterial burden, the RS ratio measures a fundamentally novel property, how drugs impact ongoing bacterial rRNA synthesis.