Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study

Reviewer #1 (Public Review):

Summary:
The study entitled "Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study" by Vijay et al. provides valuable insights into the association of rifampicin tolerance and growth fitness with isoniazid resistance among clinical isolates of M. tuberculosis. Antibiotic tolerance in M. tuberculosis is an important topic since it contributes to the lengthy and complicated treatment required to cure tuberculosis disease and may portend the emergence of antibiotic resistance. The authors found that rifampicin tolerance was correlated with bacterial growth, rifampicin minimum inhibitory concentrations, and isoniazid-resistance mutations.

Strengths:
The large number of clinical isolates evaluated and their longitudinal nature during treatment for TB (including exposure to rifampin) are strengths of the study.

Weaknesses:
Some of the methodologies are not well explained or justified and the association of antibiotic tolerance with growth rate is not a novel finding. In addition, the molecular mechanisms underlying rifampicin tolerance only in rapidly growing isoniazid-resistant isolates have not been elucidated and the potential implications of these findings for clinical management are not immediately apparent.

Reviewer #2 (Public Review):

Summary:
This study by Vijay and colleagues addresses a clinically important, and often overlooked aspect of Tb treatment. Detecting for variations in the level of antibiotic tolerance amongst otherwise antibiotic-susceptible isolates is difficult to routinely screen for, and consequently not performed. The authors, present a convincing argument that indeed, there is significant variation in the susceptibility of isoniazid-resistant strains to killing by rifampicin, in some cases at the same tolerance levels as bona fide resistant strains. On the whole, the study is easy to follow and the results are justified. This work should be of interest to the wider TB community at both a clinical and basic level.

Weaknesses:
The manuscript is long, repetitive in places, and the figures could use some amending to improve clarity (this could be a me-specific issue as they look ok on my screen, yet the colour is poor when printed).

It would have been great to have seen some correlation between increased rifampicin tolerance and treatment outcome, although I'm not sure if this data is available to the researchers. I agree with the researchers the use of a single media condition is a limitation. However, this is true of a lot of studies.

Reviewer #3 (Public Review):

Summary:
The authors have initiated studies to understand the molecular mechanisms underlying the devolvement of multi-drug resistance in clinical Mtb strains. They demonstrate the association of isoniazid-resistant isolates by rifampicin treatment supporting the idea that selection of MDR is a microenvironment phenomenon and involves a group of isolates.

Strengths:
The methods used in this study are robust and the results support the authors' claims to a major extent.

Weaknesses:
The manuscript needs a thorough vetting of the language. At present, the language makes it very difficult to comprehend the methodology and results.