Development of an In Vitro Assay for Detection of Drug-Induced Resuscitation-Promoting-Factor-Dependent Mycobacteria

Abstract

Tuberculosis is a major infectious disease that requires prolonged chemotherapy with a combination of four drugs. Here we present data suggesting that treatment of Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium smegmatis, a model organism widely used for the screening of antituberculosis agents, with first-line drugs resulted in the generation of substantial populations that could be recovered only by the addition of a culture supernatant from growing mycobacteria. These bacilli failed to grow in standard media, resulting in significant underestimation of the numbers of viable mycobacteria in treated samples. We generated M. smegmatis strains overexpressing M. tuberculosis resuscitation-promoting factors (Rpfs) and demonstrated their application for the detection of Rpf-dependent mycobacteria generated after drug exposure. Our data offer novel opportunities for validation of the sterilizing activity of antituberculosis agents.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

“…Other reported in vitro models for producing DD Mtb include hypoxia and starvation with antibiotic exposure (19), potassium depletion (41), exposure of log-phase cells to antibiotics (42), and gradual acidification (43). It is not clear why we did not succeed in producing DD Mtb with methods like these.…”

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112

“…While there are many outstanding questions on the precise mechanisms of PknB-mediated regulation of gene expression and Lsr2 binding to DNA, our findings provide a functional link between serine/threonine protein kinase signalling and transcriptional regulatory pathways that enable Mtb to survive the varied environments encountered during infection. Bacterial growth was measured by absorbance at 580nm, or by colony-forming unit (CFU) counting on 7H10 agar (Becton, Dickinson and Company), or by most probable number (MPN) counting using established protocols (Loraine et al, 2016) and the MPN calculator program (Jarvis et al, 2010). Escherichia coli OverExpress™ C41(DE3) and DH5α were grown in Lysogeny broth.…”

“…SMM comprised of 0.3 M sucrose, 20 mM MgSO 4 , 0.1% Tween 80 (w/v), 10% (v/v) ADC in standard 7H9 broth. Bacterial growth was measured by absorbance at 580nm, or by colony‐forming unit (CFU) counting on 7H10 agar (Becton, Dickinson and Company), or by most probable number (MPN) counting using established protocols (Loraine et al , ) and the MPN calculator program (Jarvis et al , ). Escherichia coli OverExpress™ C41(DE3) and DH5α were grown in Lysogeny broth.…”

Sub-minimum inhibitory concentration of rifampin: a potential risk factor for resuscitation of Mycobacterium tuberculosis

“…Loraine et al recently showed that Rpf overexpression rescues non-culturable mycobacteria, generated by treatment with first-line antimicrobial agents in vitro . In addition, Loraine et al proved that the RpfD-overexpressing M. smegmatis strains showed consistently higher resuscitation, compared to the other Rpfs [ 24 ]. Interestingly, rpfD was the most prevalent (66.66%) and overexpressed gene in our study (Table 2 ).…”