Rapid, accurate, and reproducible

As one of the deadliest infectious diseases in the world, tuberculosis is responsible for millions of new cases and deaths reported annually. The rise of drug-resistant tuberculosis, particularly resistance to first-line treatments like rifampicin, presents a critical challenge for global health, which complicates the treatment strategies and calls for effective diagnostic and predictive tools. In this study, we apply an ensemble-based molecular dynamics computer simulation method, TIES_PM, to estimate the binding affinity through free energy calculations and predict rifampicin resistance in RNA polymerase. By analyzing 61 mutations, including those in the rifampicin resistance-determining region, TIES_PM produces reliable results in good agreement with clinical reference and identifies abnormal data points indicating alternative mechanisms of resistance. In the future, TIES_PM is capable of identifying and selecting leads with a lower risk of resistance evolution and, for smaller proteins, it may systematically predict antibiotic resistance by analyzing all possible codon permutations. Moreover, its flexibility allows for extending predictions to other first-line drugs and drug-resistant diseases. TIES_PM provides a rapid, accurate, low-cost, and scalable supplement to current diagnostic pipelines, particularly for drug resistance screening in both research and clinical domains.IMPORTANCEAntimicrobial resistance (AMR), a global threat, challenges early diagnosis and treatment of tuberculosis (TB). This study employs TIES_PM, a free-energy calculation method, to efficiently predict AMR by quantifying how mutations in bacterial RNA polymerase (RNAP) affect rifampicin (RIF) binding. On simulating 61 clinically observed mutations, the results align with WHO classifications and reveal ambiguous cases, suggesting alternative resistance mechanisms. Each mutation requires ~5 h, offering rapid, cost-effective predictions. An ensemble approach ensures statistical robustness. TIES_PM can be extended to smaller proteins for systematic codon permutation analysis, enabling comprehensive antibiotic resistance prediction, or adapted to identify low-resistance-risk drug leads. It also applies to other TB drugs and resistant pathogens, supporting personalized therapy and global AMR surveillance. This work provides novel tools to refine resistance mutation databases and phenotypic classification standards, enhancing early diagnosis while advancing translational research and infectious disease control.