Genome sequence comparisons of serial multi-drug-resistant Mycobacterium tuberculosis isolates over 21 years of infection in a single patient

We report a case of chronic pulmonary multi-drug-resistant tuberculosis. Despite 14 years of treatment, Mycobacterium tuberculosis was persistently isolated from sputum. Following treatment cessation the patient remained well, although M. tuberculosis was isolated from sputum for a further 8 years. Genome sequencing of eight serial M. tuberculosis isolates cultured between 1991 and 2011 revealed 17 mutations (0.8 mutations per genome year- 1). Eight of these were persisting mutations and only two mutations were detected in the 7 years following cessation of treatment in 2004. In four isolates there were mixed alleles, suggesting the likely presence of bacterial subpopulations. The initial 1991 isolate demonstrated genotypic resistance to isoniazid (katG W91R), rifampicin (rpoB S531L), ethambutol (embB M306V), streptomycin (gidB L16R), quinolones (gyrA S95T) and P-aminosalicylic acid (thyA T202A). Subsequent resistance mutations developed for pyrazinamide (pncA I31F) and ethionamide (ethA frameshift). Such information might have been instructive when developing a treatment regimen. In retrospect and with the benefit of high-resolution genomic hindsight we were able to determine that the patient received only one or two active anti-tuberculous agents for most of their treatment. Additionally, mutations in bacA and Rv2326c were detected, which may have contributed to the persistent but mild disease course. BacA is likely to be associated with maintenance of chronic infection and Rv2326c with a decreased bacterial metabolic state. These results expand our understanding of M. tuberculosis evolution during human infection and underline the link between antibiotic resistance and clinical persistence.