Rifampicin tolerance and growth fitness among isoniazid-resistant clinicalMycobacterium tuberculosisisolates: an in-vitro longitudinal study

Antibiotic tolerance inMycobacterium tuberculosisleads to less effective bacterial killing, poor treatment responses and resistant emergence. Therefore, we investigated the rifampicin tolerance ofM. tuberculosisisolates, with or without pre-existing isoniazid-resistance. We determined thein-vitrorifampicin survival fraction by minimum duration of killing assay in isoniazid susceptible (IS, n=119) and resistant (IR, n=84)M. tuberculosisisolates. Then we correlated the rifampicin tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. The longitudinal IR isolates collected from patients were analyzed for changes in rifampicin tolerance and associated emergence of genetic variants. The median duration of rifampicin exposure reducing theM. tuberculosissurviving fraction by 90% (minimum duration of killing-MDK90) increased from 1.23 (95%CI 1.11; 1.37) and 1.31 (95%CI 1.14; 1.48) to 2.55 (95%CI 2.04; 2.97) and 1.98 (95%CI 1.69; 2.56) days, for IS and IR respectively, during 15 to 60 days of incubation. This indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log₁₀-fold survival fraction enabled classification of tolerance as low, medium or high and revealed IR association with increased tolerance with faster growth (OR=2.68 for low vs. medium, OR=4.42 for low vs. high,P-trend=0.0003). The high tolerance in IR isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Furthermore, the high rifampicin tolerant IR isolates have survival potential similar to multi-drug resistant isolates. These findings suggest that IR tuberculosis needs to be evaluated for high rifampicin tolerance to improve treatment regimen and prevent the risk of MDR-TB emergence.