Maha R Farhat1,2, Jaimie Sixsmith3, Roger Calderon4, Nathan D Hicks3, Sarah M Fortune3, Megan Murray5,6. 1. Department of Biomedical Informatics, Harvard Medical School, 10 Shattuck Street, Boston, MA, USA. 2. Division of Pulmonary and Critical Care, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, USA. 3. Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, 677 Huntington Avenue, Boston, MA, USA. 4. Socios en Salud, Calle Los Geranios 312, Lince, Peru. 5. Department of Global Health and Social Medicine, Harvard Medical School, 641 Huntington Avenue, Boston, MA, USA. 6. Division of Global Health Equity, Brigham and Women's Hospital, 75 Francis Street, Boston, MA, USA.
Abstract
OBJECTIVES: Drug-resistant TB remains a public health challenge. Rifamycins are among the most potent anti-TB drugs. They are known to target the RpoB subunit of RNA polymerase; however, our understanding of how rifamycin resistance is genetically encoded remains incomplete. Here we investigated rpoB genetic diversity and cross-resistance between the two rifamycin drugs rifampicin and rifabutin. METHODS: We performed WGS of 1003 Mycobacterium tuberculosis clinical isolates and determined MICs of both rifamycin agents on 7H10 agar using the indirect proportion method. We generated rpoB mutants in a laboratory strain and measured their antibiotic susceptibility using the alamarBlue reduction assay. RESULTS: Of the 1003 isolates, 766 were rifampicin resistant and 210 (27%) of these were rifabutin susceptible; 102/210 isolates had the rpoB mutation D435V (Escherichia coli D516V). Isolates with discordant resistance were 17.2 times more likely to harbour a D435V mutation than those resistant to both agents (OR 17.2, 95% CI 10.5-27.9, P value <10-40). Compared with WT, the D435V in vitro mutant had an increased IC50 of both rifamycins; however, in both cases to a lesser degree than the S450L (E. coli S531L) mutation. CONCLUSIONS: The observation that the rpoB D435V mutation produces an increase in the IC50 of both drugs contrasts with findings from previous smaller studies that suggested that isolates with the D435V mutation remain rifabutin susceptible despite being rifampicin resistant. Our finding thus suggests that the recommended critical testing concentration for rifabutin should be revised.
OBJECTIVES: Drug-resistant TB remains a public health challenge. Rifamycins are among the most potent anti-TB drugs. They are known to target the RpoB subunit of RNA polymerase; however, our understanding of how rifamycin resistance is genetically encoded remains incomplete. Here we investigated rpoB genetic diversity and cross-resistance between the two rifamycin drugs rifampicin and rifabutin. METHODS: We performed WGS of 1003 Mycobacterium tuberculosis clinical isolates and determined MICs of both rifamycin agents on 7H10agar using the indirect proportion method. We generated rpoB mutants in a laboratory strain and measured their antibiotic susceptibility using the alamarBlue reduction assay. RESULTS: Of the 1003 isolates, 766 were rifampicin resistant and 210 (27%) of these were rifabutin susceptible; 102/210 isolates had the rpoB mutation D435V (Escherichia coliD516V). Isolates with discordant resistance were 17.2 times more likely to harbour a D435V mutation than those resistant to both agents (OR 17.2, 95% CI 10.5-27.9, P value <10-40). Compared with WT, the D435V in vitro mutant had an increased IC50 of both rifamycins; however, in both cases to a lesser degree than the S450L (E. coliS531L) mutation. CONCLUSIONS: The observation that the rpoB D435V mutation produces an increase in the IC50 of both drugs contrasts with findings from previous smaller studies that suggested that isolates with the D435V mutation remain rifabutin susceptible despite being rifampicin resistant. Our finding thus suggests that the recommended critical testing concentration for rifabutin should be revised.
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