Study design

(A) Study design. IS – Isoniazid susceptible, IR – Isoniazid resistant, RR – Rifampicin resistant. (B) Most-probable number based rifampicin killing assay and survival fraction determination.

Rifampicin survival curve in isoniazid susceptible and resistant clinical M. tuberculosis isolates.

(A, B) The bacterial kill curve as measured by log10 survival fraction from data collected at 0, 2 and 5 days of rifampicin treatment followed by incubation for 15 and 60 days respectively. Data from individual isolates are shown as the grey dots connected by lines. Estimated mean with 95% credible interval (bold coloured line and colour shaded area respectively) of isoniazid susceptible (IS – blue, n = 119, 117 for 15 and 60 days of incubation respectively) and resistant (IR – red, n = 84, 80 for 15 and 60 days of incubation respectively) clinical M. tuberculosis isolates based on linear mixed effect model implemented in a Bayesian framework. One log10 fold or 90% reduction in survival fraction is indicated (MDK90, black horizontal line) and the mean time duration required for 90% reduction in survival (MDK90, minimum duration of killing time) at 15 and 60 days of incubation is indicated by vertical dashed lines with respective colours for IS and IR isolates.

Rifampicin survival fraction distribution in isoniazid susceptible and resistant clinical M. tuberculosis isolates.

(A) Log10 rifampicin survival fraction distribution, with median and IQR (inter quartile range), of individual isoniazid susceptible (IS, blue dots, n = 119, 117 for D5-15, and D5-60 respectively), and resistant (IR, red dots, n = 84, 80 for D5-15, D5-60 respectively) isolates for 5 days of rifampicin treatment as determined at 15 and 60 days of incubation (D5-15, D5-60 respectively). (B) Rifampicin tolerance distribution in both IS (blue dots) and IR (red dots) isolates combined together (All) was used to group them as low (< 25th percentile, n = 33, 47 for D5-15, and D5-60 respectively), medium (from 25th to 75th percentile, n = 124, 115 for D5-15, and D5-60 respectively) and high (above 75th percentile, n = 46, 35 for D5-15, and D5-60 respectively) level of rifampicin tolerance and compare it with rifampicin tolerance of MDR clinical M. tuberculosis isolates (grey dots, n = 6), after 5 days of rifampicin treatment and determined at 15 and 60 days of incubation (D5-15, D5-60 respectively). Statistical comparisons between Low, Medium, and High or MDR were made by using Wilcoxon rank-sum test. # - p-value for comparing the Low and High tolerance groups, ^ - p-value for comparing the medium and High tolerance groups.

Association of rifampicin tolerance level with isoniazid susceptibility

Correlating rifampicin survival fraction with before treatment relative growth of clinical M. tuberculosis isolates.

Log10 survival fraction at 5 days of rifampicin treatment as determined at 15 days (A) and 60 days of incubation (B), for isoniazid susceptible (IS, blue dots) and resistant (IR, red dots) isolates respectively, correlated with the log10 relative growth determined before rifampicin treatment for clinical M. tuberculosis isolates. Coefficients of linear regression for (A) IS = - 0.21 [-0.44, 0.007], P = 0.058; IR = −0.12 [-0.38, 0.14], P = 0.37, and (B) IS = 0.38 [0.15, 0.61], P = 0.0014; IR = 0.38 [0.12, 0.64], P = 0.0041. (C) Log10 distribution of relative growth with median and IQR for IS and IR clinical M. tuberculosis isolates before rifampicin treatment. Statistical comparisons between IS and IR were made by using Wilcoxon rank-sum test.

Rifampicin survival fraction distribution in isoniazid susceptible and longitudinal isoniazid resistant clinical M. tuberculosis isolates.

Log10 rifampicin survival fraction distribution, with median and IQR (inter quartile range), of individual isoniazid susceptible (IS, blue dots, n = 119, 117 for D5-15, and D5-60 respectively), and longitudinal isoniazid resistant (IR, red dots, n = 84, 80 for D5-15, D5-60 respectively) isolates for 5 days of rifampicin treatment as determined at 15 and 60 days of incubation (D5-15, D5-60 respectively) grouped based on collection time as baseline (IR-BL, n = 49), intensive phase (IR-IP, n = 14), and continuous phase and relapse (IR-CP, n = 21). Statistical comparisons between groups were made by using Krusal-Walis test.

Rifampicin tolerance of longitudinal isoniazid resistant clinical M. tuberculosis isolates from individual patients.

(A, B) Rifampicin tolerance heat map after 5 days of rifampicin treatment as determined at 15 and 60 days of incubation (D5-15, D5-60 respectively), of longitudinal isoniazid resistant clinical M. tuberculosis isolates collected from individual patients during different months of treatment and follow-up. Longitudinal isoniazid resistant clinical M. tuberculosis isolates from individual patients are grouped based on changes in rifampicin tolerance compared between initial and subsequent months of collection as decrease, un change and increase. Months (0 – 24) represent the different months the isolates were collected from patients during 8 months treatment and 24 months follow-up.

Genetic variants associated with changes in rifampicin tolerance.

Non-synonymous single nucleotide polymorphism emerging in pair-wise comparison of longitudinally collected isoniazid resistant M. tuberculosis isolates from same patient associated with increase (red), decrease (dark blue) and no change (light violet) in rifampicin tolerance phenotype at 15 and 60 days of incubation (D5-15 and D5-60 respectively). Each count represents a single independent SNP emergence event.