Precursor ribosomal RNA as a biomarker for effective mycobacterial treatment

Mycobacteria range from environmental bacteria to opportunistic and obligate pathogens. Mycobacterium tuberculosis kills ~1.5 million people each year and incidence of drug-resistant tuberculosis increases yearly. Similarly, Mycobacterium abscessus infections are rising in immunocompromised populations and treatment prospects are grim. Contributing to the incidence of drug-resistant infections is the lack of quantifiable data on drug regimen efficacy. This document describes a novel biomarker and associated metrics for use in evaluating mycobacterial drugs and drug regimens at all stages of development. The ribosomal RNA (rRNA) synthesis ratio measures ongoing rRNA synthesis by comparing precursor rRNA species to total 23S rRNA to determine ongoing ribosomal synthesis as a correlative measure of bacterial replication. With this biomarker, we can determine the physiologic state of bacteria under drug treatment, gleaning information on how the bacteria respond to drug pressure in addition to the traditional metrics of bacterial burden, which are known to not correlate with relapse probability.In both in vitro and in vivo M. tuberculosis models, drugs that shorten the time of treatment needed to achieve cure without relapse of disease – treatment-shortening drugs – reduced the rRNA synthesis ratio rapidly and profoundly. Non-treatment-shortening drugs only modestly decreased the rRNA synthesis ratio. These data indicate that treatment-shortening drugs stop rRNA synthesis and likely stop replication, whereas drugs that do not shorten treatment allow for continued bacterial replication even under lethal drug pressure. The rRNA synthesis ratio also allows for a more nuanced analysis of drug activity compared to measures of burden. In both M. tuberculosis and M. abscessus, treatment with bedaquiline resulted in complete inhibition of bacterial replication. Analysis of the rRNA synthesis ratio indicated that while bedaquiline initially reduced the ratio in both bacteria, the ratio slowly rebounded over time in M. abscessus. Only when M. abscessus was treated with bedaquiline and clofazimine in combination was the rRNA synthesis ratio kept sustainably low. The bacterial burden of M. abscessus treated with bedaquiline compared to bedaquiline in combination with clofazimine was identical, further highlighting the difference between analysis of physiologic state and burden. The data herein indicate that effective treatment in mycobacterial infections likely should consist of at least one drug that stops replication in addition to one or more potent bactericidal drugs. The rRNA synthesis ratio provides crucial information that adds to traditional metrics of bacterial burden and has great potential as a metric for drug development and surrogate marker for relapse potential in animal infection models. The rRNA synthesis ratio is also being developed as a biomarker of treatment efficacy in humans to help inform clinical trials and for possible use in adaptive clinical trial design and stratified patient care.