Aims
The hollow‐fibre system for tuberculosis (HFS‐TB) is a preclinical model qualified by the European Medicines Agency to underpin the anti‐TB drug development process. It can mimic in vivo ...pharmacokinetic (PK)–pharmacodynamic (PD) attributes of selected antimicrobials, which could feed into in silico models to inform the design of clinical trials. However, historical data and published protocols are insufficient and omit key information to allow experiments to be reproducible. Therefore, in this work, we aim to optimize and standardize various HFS‐TB operational procedures.
Methods
First, we characterized bacterial growth dynamics with different types of hollow‐fibre cartridges, Mycobacterium tuberculosis strains and media. Second, we mimicked a moxifloxacin PK profile within hollow‐fibre cartridges, in order to check drug–fibres compatibility. Lastly, we mimicked the moxifloxacin total plasma PK profile in human after once daily oral dose of 400 mg to assess PK–PD after different sampling methods, strains, cartridge size and bacterial adaptation periods before drug infusion into the system.
Results
We found that final bacterial load inside the HFS‐TB was contingent on the studied variables. Besides, we demonstrated that drug–fibres compatibility tests are critical preliminary HFS‐TB assays, which need to be properly reported. Lastly, we uncovered that the sampling method and bacterial adaptation period before drug infusion significantly impact actual experimental conclusions.
Conclusion
Our data contribute to the necessary standardization of HFS‐TB experiments, draw attention to multiple aspects of this preclinical model that should be considered when reporting novel results and warn about critical parameters in the HFS‐TB currently overlooked.
Bedaquiline (BDQ) is a recently approved antibiotic for the treatment of multidrug-resistant tuberculosis, but its potential against slow-growing mycobacteria (SGM) is still unknown. The objective of ...this study was to determine the in vitro activity of BDQ on SGM by assessing their MIC and minimal bactericidal concentration (MBC). The MIC of BDQ against 17 clinical isolates including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium kansasii and Mycobacterium simiae species was determined by the resazurin microtitre assay and the MBC by the c.f.u. determination on 7H10 agar plates. BDQ has a bacteriostatic activity on all SGM tested with a MIC range from 0.03 to 0.007 µg ml
and surprisingly a good bactericidal activity on the majority of the isolates tested with an MBC of 1-2 µg ml
. Based on these preliminary results BDQ seems to be very promising for treatment of diseases caused by SGM.
Bedaquiline (BDQ) has been proven to be effective in the treatment of multidrug-resistant tuberculosis. We hypothesized that BDQ could be a potential agent to treat nontuberculous mycobacterial (NTM) ...infection. The objective of this study was to evaluate the in vitro activity of BDQ against rapidly growing mycobacteria by assessing the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 18 NTM strains. For MIC determination we performed the resazurin microtitre assay broth dilution, and for the MBC the c.f.u. was determined. BDQ exhibited a strong inhibitory effect against most NTM tested; however, for some NTM strains the MBC was significantly higher than the MIC. A new finding is that Mycobacterium flavescens has a mutation in the gene atpE associated with natural resistance to BDQ. These preliminary promising results demonstrate that BDQ could be potentially useful for the treatment of NTM.
Strong evidence supports the idea that fatty acids rather than carbohydrates are the main energy source of Mycobacterium tuberculosis during infection and latency. Despite that important role, a ...complete scenario of the bacterium's metabolism when lipids are the main energy source is still lacking. Here we report the development of an in vitro model to analyze adaptation of M. tuberculosis during assimilation of long-chain fatty acids as sole carbon sources. The global lipid transcriptome revealed a shift toward the glyoxylate cycle, the overexpression of main regulators whiB3, dosR, and Rv0081, and the increased expression of several genes related to reductive stress. Our evidence showed that lipid storage seems to be the selected mechanism used by M. tuberculosis to ameliorate the assumed damage of reductive stress and that concomitantly the bacilli acquired a slowed-growth and drug-tolerant phenotype, all characteristics previously associated with the dormant stage. Additionally, intergenic regions were also detected, including the unexpected upregulation of tRNAs that suggest a new role for these molecules in the acquisition of a drug-tolerant phenotype by dormant bacilli. Finally, a set of lipid signature genes for the adaptation process was also identified. This in vitro model represents a suitable condition to illustrate the participation of reductive stress in drugs' activity against dormant bacilli, an aspect scarcely investigated to date. This approach provides a new perspective to the understanding of latent infection and suggests the participation of previously undetected molecules.
Mycobacterium tuberculosis establishes long-lasting highly prevalent infection inside the human body, called latent tuberculosis. The known involvement of fatty acids is changing our understanding of that silent infection; however, question of how tubercle bacilli globally adapt to a lipid-enriched environment is still an unanswered. With the single change of providing fatty acids as carbon sources, the bacilli switch on their program related to dormant stage: slowed growth, accumulation of lipid bodies, and development of drug tolerance. In this stage, unexpected and previously unknown participants were found to play putatively important roles during the process. For the first time, this work compares the global transcriptomics of bacteria by using strand-specific RNA sequencing under two different growth conditions. This study suggests novel targets for the control of tuberculosis and provides a new straightforward in vitro model that could help to test the activity of drugs against dormant bacilli from a novel perspective.
Although tuberculosis treatment is dependent on drug-susceptibility testing (DST) and molecular drug-resistance detection, treatment failure and relapse remain a challenge. This could be partially ...due to the emergence of antibiotic-tolerant dormant mycobacteria, where host lipids have been shown to play an important role. This study evaluated the susceptibility of Mycobacterium tuberculosis to two antibiotic combinations - rifampicin, moxifloxacin, amikacin and metronidazole (RIF-MXF-AMK-MTZ), and rifampicin, moxifloxacin, amikacin and pretomanid (RIF-MXF-AMK-PA) - in a lipid-rich dormancy model. Although their effectiveness in in vitro cultures with dextrose as a carbon source has been proved, we observed that none of the antibiotic mixtures were bactericidal in the presence of lipids. The presence of lipids may confer tolerance to M. tuberculosis against the mixture of antibiotics tested and such tolerance could be even higher during the dormant stages. The implementation of lipids in DST on clinical isolates could potentially lead to a better treatment strategy.
Abstract It is known that cholesterol plays a key role for Mycobacterium tuberculosis ( Mtb ) adaptation and survival within the host, thus contributing to the establishment of dormancy. It has been ...extensively demonstrated that fatty acids are the main energy source of Mtb during infection and dormancy, and it has been proposed that these molecules are implicated in reactivation of bacilli from a dormant state. We used in vitro models to analyze Mtb gene expression during dormancy and reactivation when fatty acids and cholesterol are the unique carbon source in the media. Our results suggest that cholesterol might function as a signal to trigger Mtb expression of some genes required for stress protection earlier than the one induced by fatty acids alone, indicating that cholesterol is very favorable for its development. This process is so conducive that cholesterol-adapted bacilli can reactivate their growth after NRP2 dormancy state even 10 min post ventilation. Thus, we hypothesize that cholesterol is not only involved in Mtb dormancy but that it also plays a critical role for favorable and almost immediate reactivation from an in vitro long-lasting dormant state induced by hypoxia.
Tuberculosis (TB) remains as one of the leading causes of morbidity and mortality among infectious diseases worldwide. Although lipids (mainly fatty acids and cholesterol) have been reported to play ...an important role during active and latent infection of M. tuberculosis, there are other molecular aspects of bacterial response to those substrates that are not fully understood, involving gene regulation background. This review highlights recent insights on pathogen gene expression: regulation during its active growth, during survival in presence of lipids and under variable hostile host microenvironments. We also propose several application options of this knowledge that may contribute for improved TB control.
•The role of lipids in the survival and adaptation of M. tuberculosis within different host microenvironments are detailed.•The role of transcriptional factors as modulators that drive lipid metabolism of bacilli for survival is critically valued.•The importance of investigating ncRNAs, and genes related to lipid metabolism during the TB infection is examined.•Some approaches for TB control based on genetic response of M. tuberculosis to a lipid-rich environment are discussed.
Nontuberculous mycobacteria (NTM) are environmental opportunistic pathogens found in natural and human-engineered waters, including drinking water distribution systems and household plumbing. This ...pilot study examined the frequency of occurrence of NTM in household potable water samples in Mexico City. Potable water samples were collected from the "main house faucet" and kitchen faucet. The presence of aerobic-mesophilic bacteria (AMB), total coliforms (TC), fecal coliforms (FC) and NTM species were determined. Mycobacteria species were identified by PCR restriction enzyme pattern analysis (PRA) of the 65-kDa heat shock protein gene (hsp65) and sequencing of the hypervariable region 2 (V2) of the 16S rRNA gene and of the rpoB gene.
AMB (<100 CFU/ml) were present in 118 out of 120 samples; only two samples were outside guidelines ranges (>100 CFU/ml). TC and FC were detected in four and one samples, respectively. NTM species were recovered from 16% samples (19/120) and included M. mucogenicum (nine), M. porcinum (three), M. avium (three), M. gordonae (one), M. cosmeticum (one), M. fortuitum (one), and Mycobacterium sp (one). All household water samples that contained NTM complied with the standards required to grade the water as "good quality" potable water.
Household potable water may be a potential source of NTM infection in Mexico City.
Tuberculosis (TB) is the historical leading cause of death by a single infectious agent. The European Regimen Accelerator for Tuberculosis (ERA4TB) is a public-private partnership of 30+ institutions ...with the objective to progress new anti-TB regimens into the clinic. Thus, robust and replicable results across independent laboratories are essential for reliable interpretation of treatment efficacy. A standardization workgroup unified in vitro protocols and data reporting templates. Time-kill assays provide essential input data for pharmacometric model-informed translation of single agents and regimens activity from in vitro to in vivo and the clinic. Five conditions were assessed by time-kill assays in six independent laboratories using four bacterial plating methods. Baseline bacterial burden varied between laboratories but variability was limited in net drug effect, confirming 2.5 μL equally robust as 100 μL plating. This exercise establishes the foundations of collaborative data generation, reporting, and integration within the overarching Antimicrobial Resistance Accelerator program.
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•Best collaboration practices are shared on a large consortium initiative (ERA4TB)•Robust standardized time kill assay protocol for Mycobacterium tuberculosis•Variability—inherent to the assay—was successfully quantified and minimized•An innovative 2.5 μL plating technique performed similar to the 100 μL standard
Treatment; Molecular modeling; Biochemical assay
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