Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study
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eLife assessment
This valuable study demonstrates that there is significant variation in the susceptibility of isoniazid-resistant Mycobacterium tuberculosis clinical isolates to killing by rifampicin, in some cases at the same tolerance levels as bona fide resistant strains. The evidence provided is solid, with no clear genetic marker for increased tolerance, suggesting that there may be multiple routes to achieving this phenotype. The work will be of interest to infectious disease researchers.
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Abstract
Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. There is limited understanding of antibiotic tolerance in clinical isolates of M. tuberculosis . Therefore, we investigated the rifampicin tolerance of M. tuberculosis isolates, with or without pre-existing isoniazid-resistance. In-vitro rifampicin survival fractions determined by minimum duration of killing assay in isoniazid susceptible (n=119) and resistant (n=84) M. tuberculosis isolates. Rifampicin tolerance was correlated with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. The longitudinal isoniazid-resistant isolates were analyzed for rifampicin tolerance based on collection time from patients and associated emergence of genetic variants. The median duration of rifampicin exposure reducing the M. tuberculosis surviving 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 respectively. Increase in MDK90 time indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log 10 -fold survival fraction enabled classification of tolerance as low, medium or high and revealed isoniazid-resistance 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 longitudinal isoniazid-resistant isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Our study identifies a range of rifampicin tolerance and reveals that isoniazid resistance is associated with higher tolerance with growth fitness. Furthermore, rifampicin treatment may select isoniazid-resistant isolate microvariants with higher rifampicin tolerance, with survival potential similar to multi-drug resistant isolates. These findings suggest that isoniazid-resistant tuberculosis needs to be evaluated for rifampicin tolerance or needs further improvement in treatment regimen.
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eLife assessment
This valuable study demonstrates that there is significant variation in the susceptibility of isoniazid-resistant Mycobacterium tuberculosis clinical isolates to killing by rifampicin, in some cases at the same tolerance levels as bona fide resistant strains. The evidence provided is solid, with no clear genetic marker for increased tolerance, suggesting that there may be multiple routes to achieving this phenotype. The work will be of interest to infectious disease researchers.
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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 …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 …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.
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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. -
