Clofazimine as a substitute for rifampicin improves efficacy of Mycobacterium avium pulmonary disease treatment in the hollow-fiber model.

Mycobacterium avium complex pulmonary disease is treated with an azithromycin, ethambutol, and rifampicin regimen, with limited efficacy. The role of rifampicin is controversial due to inactivity, adverse effects, and drug interactions. Here, we evaluated the efficacy of clofazimine as a substitute for rifampicin in an intracellular hollow-fiber infection model. THP-1 cells, which are monocytes isolated from peripheral blood from an acute monocytic leukemia patient, were infected with M. avium ATCC 700898 and exposed to a regimen of azithromycin and ethambutol with either rifampicin or clofazimine. Intrapulmonary pharmacokinetic profiles of azithromycin, ethambutol, and rifampicin were simulated. For clofazimine, a steady-state average concentration was targeted. Drug concentrations and bacterial densities were monitored over 21 days. Exposures to azithromycin and ethambutol were 20%-40% lower than targeted but within clinically observed ranges. Clofazimine exposures were 1.7 times higher than targeted. Until day 7, both regimens were able to maintain stasis. Thereafter, regrowth was observed for the rifampicin-containing regimen, while the clofazimine-containing regimen yielded a 2 Log10 colony forming unit (CFU) per mL decrease in bacterial load. The clofazimine regimen also successfully suppressed the emergence of macrolide tolerance. In summary, substitution of rifampicin with clofazimine in the hollow-fiber model improved the antimycobacterial activity of the regimen. Clofazimine-containing regimens merit investigation in clinical trials.

A Mycobacterium tuberculosis-Infected Patient Who Could Not Tolerate Oral Intake Successfully Treated Using an Intravenous Tedizolid-Containing Regimen.

BACKGROUND Mycobacterium tuberculosis (M. tuberculosis) is usually treated by oral antimycobacterial agents, including rifampicin, ethambutol, and pyrazinamide, but the treatment regimen with intravenous and/or intramuscular antimycobacterial agents for patients who cannot take medications orally remains unclear. CASE REPORT A 77-year-old man with chronic renal failure had an esophageal-skin fistula after he had surgeries for removal of esophageal and gastric cancers and reconstruction using jejunum, and he showed a cavity, tree-in-bud formation, and pleural effusions in his left upper lung fields on his chest X-ray after treatment of cellulitis and bacteremia/candidemia by meropenem, teicoplanin, and micafungin. M. tuberculosis was isolated from his sputum and exudate fluid from the reconstructed esophageal-skin fistula. Although he could not take antimycobacterial agents orally, treatment was started with intravenous agents combining levofloxacin (LVFX) every other day, isoniazid (INH), and linezolid (LZD). However, his platelets were decreased 21 days after treatment started, and it was thought to be an adverse effect of LZD and/or INH. After changing LZD to tedizolid (TZD), in addition to changing from INH to intramuscular streptomycin twice per week, his platelet counts increased. Intravenous TZD could be continued, and it maintained his condition without exacerbations of thrombocytopenia and renal failure. The M. tuberculosis disappeared, and the abnormal chest X-ray shadows were improved 2 months after the start of treatment. CONCLUSIONS Administration of intravenous TZD, in addition to intravenous LVFX and intramuscular SM in combination, might be a candidate regimen for M. tuberculosis patients who cannot take oral medications.

[Action Mechanism of Ethambutol Tablets on Pulmonary Tuberculosis Rat Model Based on Janus Kinase/Signal Transducer and Activator of Transcription Signaling Pathway].

Objective To explore the therapeutic effect of ethambutol tablets (EMB) on pulmonary tuberculosis (PTB) in rats and whether the action mechanism of EMB is related to Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Methods Sixty SD rats were assigned into a control group,a PTB group,a PTB+EMB group (30 mg/kg),and a PTB+EMB+Colivelin (JAK/STAT pathway activator) group (30 mg/kg+1 mg/kg) via the random number table method,with 15 rats in each group.The rats in other groups except the control group were injected with 0.2 ml of 5 mg/ml Mycobacterium tuberculosis suspension to establish the PTB model.After the modeling,the rats were administrated with corresponding drugs for 4 consecutive weeks (once a day).On days 1,14,and 28 of administration,the body weights of rats were measured and the Mycobacterium tuberculosis colonies were counted.Hematoxylin-eosin staining was carried out to detect the pathological changes in the lung tissue.Enzyme-linked immunosorbent assay was employed to measure the levels of interleukin(IL)-6,tumor necrosis factor-α (TNF-α),IL-1ß,and interferon-γ (IFN-γ) in the serum.Flow cytometry was used to determine the levels of T lymphocyte subsets CD3+,CD4+,CD8+,and CD4+/CD8+.The 16S rRNA sequencing was performed to detect the relative abundance of the intestinal microorganisms.Western blotting was employed to determine the expression of the proteins in the JAK/STAT pathway. Results Compared with the control group,the modeling of PTB reduced the rat body weight (on days 14 and 28),increased Mycobacterium tuberculosis colonies,caused severe pathological changes in the lung tissue,and elevated the levels of IL-6,TNF-α,and IL-1ß in serum and CD8+.Moreover,the modeling increased the relative abundance of Bacteroides,Peptococcus,Clostridium,Actinomyces,Lactobacillus,Verrucomicrobium,and Veillonella in the intestine,up-regulated the protein levels of phosphorylated JAK2 and phosphorylated STAT3 in the lung tissue,and lowered the levels of CD3+,CD4+,CD4+/CD8+,and IFN-γ levels (all P<0.001).Compared with the PTB group,PTB+EMB increased the rat body weight (on days 14 and 28),reduced Mycobacterium tuberculosis colonies,alleviated the pathological damage in lung tissue,lowered the levels of IL-6,TNF-α,and IL-1ß in serum and CD8+.Moreover,the treatment decreased the relative abundance of Bacteroides,Peptococcus,Clostridium,Actinomyces,Lactobacillus,Verrucomicrobium,Veillonella in the intestine,down-regulated the protein levels of phosphorylated JAK2 and phosphorylated STAT3 in the lung tissue,and elevated the levels of CD3+,CD4+,CD4+/CD8+,and IFN-γ (all P<0.001).Colivelin weakened the alleviation effect of EMB on PTB (all P<0.001). Conclusion EMB can inhibit the JAK/STAT signaling pathway to alleviate the PTB in rat.

The Effect of Citicoline on Ethambutol Optic Neuropathy: Histopathology and Immunohistochemistry Analysis of Retina Ganglion Cell Damage Level in Rat Model.

Purpose: Ethambutol therapy in certain doses and period can cause bilateral ocular intoxication. There is no definitive therapy that has been found to prevent damage to retina neuronal cells in ethambutol optic neuropathy (EON) cases. Citicoline is thought to have a potential effect to maintain retinal neuron cells. This study aimed to analyze the effect of citicoline on damaged rat ganglion cells in EON. Methods: An experimental study of 15 Wistar rats was divided into 3 groups: the nontreatment group (A), the ethambutol (35 mg/kg/day) group (B), and the ethambutol (35 mg/kg/day) and citicoline (1 g/kg/day) group (C). Groups B and C were given treatment orally for 30 days, then a histopathology examination was performed to analyze retinal ganglion cell (RGC) density, and immunohistochemistry to assess bcl-2 and caspase-3 expression. Results: RGC density of rat with ethambutol intoxication that received citicoline was higher than those who did not get citicoline (P < 0.001). The rat retina ganglion layer without citicoline administration is thicker than the one with citicoline, the increase in thickness is due to the formation of vacuoles in the cytoplasm of ganglion cells. Rat with citicoline obtained higher bcl-2 ganglion expression, and lower caspase-3 expression compared with rat without citicoline. Conclusions: The ganglion cells damage process caused by EON can be suppressed by citicoline administration. It was proven by analyzing RGC density, ganglion layer thickness, and expression level of bcl-2 and caspase-3 on rat model.

Identification of novel and potential PPARγ stimulators as repurposed drugs for MCAO associated brain degeneration.

Peroxisome proliferator-activated receptor-gamma (PPARγ) has been shown to have therapeutic promise in the treatment of ischemic stroke and is supported by several studies. To identify possible PPARγ activators, the current study used an in silico technique in conjunction with molecular simulations and in vivo validation. FDA-approved drugs were evaluated using molecular docking to determine their affinity for PPARγ. The findings of molecular simulations support the repurposing of rabeprazole and ethambutol for the treatment of ischemic stroke. Adult Sprague Dawley rats were subjected to transient middle cerebral artery occlusion (t-MCAO). Five groups were made as a sham-operated, t-MCAO group, rabeprazole +t-MCAO, ethambutol +t-MCAO, and pioglitazone +t-MCAO. The neuroprotective effects of these drugs were evaluated using the neurological deficit score and the infarct area. The inflammatory mediators and signaling transduction proteins were quantified using Western blotting, ELISA, and immunohistochemistry. The repurposed drugs mitigated cerebral ischemic injury by PPARγ mediated downregulation of nods like receptor protein 3 inflammasomes (NLRP3), tumor necrosis factor-alpha (TNF-α), cyclooxygenase 2 (COX-2), nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-kB), and c-Jun N-terminal kinase (p-JNK). Our data demonstrated that rabeprazole and ethambutol have neuroprotective potential via modulating the cytoprotective stress response, increasing cellular survival, and balancing homeostatic processes, and so may be suitable for future research in stroke therapy.

First-line anti-tuberculosis drug resistance trends of Mycobacterium tuberculosis complex isolates. A tertiary hospital study in Turkey.

INTRODUCTION: Tuberculosis (TB) is continuing to be a important public health problem in the undeveloped countries. Drug sensitivity rate should be monitored for the effective treatment and control in the TB. The aim of this study was to determine the rate of resistance to first line TB drugs in the Mycobacterium tuberculosis complex isolates. MATERIALS AND METHODS: During one-year period, M. tuberculosis complex was isolated in the 1193 samples from 974 patients in the Mycobacterial Laboratory of Yedikule Chest Diseases and Chest Surgery Education and Research Hospital, Istanbul, Turkey. The majority of samples isolated in the M. tuberculosis complex were sputum (n= 897, 92.1%). Anti-TB drug susceptibility testing was performed with Mycobacterium Growth Indicator Tube 960 system. RESULT: Two hundred and sixty isolat (26.7%) were resistant to at least one of the four first-line anti-TB drugs tested. One hundred ninety seven isolates were resistances to isoniazid (20.2%); 82 to rifampin (8.4%), 63 to ethambutol (6.5%) and 140 to streptomycin (14.4%). Of the 197 isoniazid-resistant isolates, 89 (45.2%) isolates was only isoniazid-resistance, only rifampin-resistance were found 15.9% (n= 13), ethambutol 7.9% (n= 5) and streptomycin 30.7% (n= 43). There were 48 (4.9%) isolates with two drugresistance, 22 (2.3%) isolates with three drug-resistance, and 42 (4.3%) isolates with four drug-resistance. The multidrug resistance rate was 7% (68 of 974). There was no relationship with between the frequency of TB drug resistance and gender or age. The isoniazid--resistance and streptomycin-resistance were seen to tend to increase if together considered the results of this study with outcomes of previously reported studies from Turkey in the 1998-2003, 2004-2007 and 2008-2010 years. CONCLUSIONS: Monitoring of drug susceptibility test results can contribute to the management of TB treatment and increase treatment success. Isoniazid-resistance and streptomycin-resistance tend to increase in Turkey. Further clinical studies are needed to investigate regional and global factors affecting the development of resistance to first-line TB drugs.

Novel 4-methoxynaphthalene-N-acylhydrazones as potential for paracoccidioidomycosis and tuberculosis co-infection.

Aim: 17 new 4-methoxynaphthalene-N-acylhydrazones were synthesized in order to evaluate their biological action against important pathogens. Methods: In vitro susceptibility assays of compounds were performed against Paracoccidioidesbrasiliensis and Mycobacterium tuberculosis. Results: Compounds 4a, 4b and 4k were the most potent against P. brasiliensis, two with minimum inhibitory concentrations of ≤1 µg ml-1 and exhibited pharmacological synergy with amphotericin B. The compounds also showed activity against M. tuberculosis, with 4c and 4k being the more promising. Compound 4k showed good synergistic antimycobacterium activity with ethambutol. None of the compounds tested showed toxicity. Conclusion: We highlight the compound 4k, as a potential agent for the treatment of patients co-infected with paracoccidioidomycosis and tuberculosis.

Modeling Host-Pathogen Interaction to Elucidate the Metabolic Drug Response of Intracellular Mycobacterium tuberculosis.

Little is known about the metabolic state of Mycobacterium tuberculosis (Mtb) inside the phagosome, a compartment inside phagocytes for killing pathogens and other foreign substances. We have developed a combined model of Mtb and human metabolism, sMtb-RECON and used this model to predict the metabolic state of Mtb during infection of the host. Amino acids are predicted to be used for energy production as well as biomass formation. Subsequently we assessed the effect of increasing dosages of drugs targeting metabolism on the metabolic state of the pathogen and predict resulting metabolic adaptations and flux rerouting through various pathways. In particular, the TCA cycle becomes more important upon drug application, as well as alanine, aspartate, glutamate, proline, arginine and porphyrin metabolism, while glycine, serine, and threonine metabolism become less important. We modeled the effect of 11 metabolically active drugs. Notably, the effect of eight could be recreated and two major profiles of the metabolic state were predicted. The profiles of the metabolic states of Mtb affected by the drugs BTZ043, cycloserine and its derivative terizidone, ethambutol, ethionamide, propionamide, and isoniazid were very similar, while TMC207 is predicted to have quite a different effect on metabolism as it inhibits ATP synthase and therefore indirectly interferes with a multitude of metabolic pathways.

The Combination Rifampin-Nitazoxanide, but Not Rifampin-Isoniazid-Pyrazinamide-Ethambutol, Kills Dormant Mycobacterium tuberculosis in Hypoxia at Neutral pH.

The activities of rifampin, nitazoxanide, PA-824, and sutezolid were tested against dormant Mycobacterium tuberculosis under conditions mimicking caseous granulomas (hypoxia at pH 7.3) in comparison with those of the combination rifampin-isoniazid-pyrazinamide-ethambutol (R-I-Z-E), which is used for human therapy. Mycobacterial viability was monitored by CFU and regrowth in MGIT 960. As shown by lack of regrowth in MGIT, rifampin-nitazoxanide-containing combinations, but not R-I-Z-E, killed dormant cells in 28 to 35 days. These observations might be important in designing new tuberculosis therapies.

New insights on Ethambutol Targets in Mycobacterium tuberculosis.

BACKGROUND: In recent years, very few effective drugs against Mycobacterium tuberculosis have emerged, which motivates the research with drugs already used in the treatment of tuberculosis. Ethambutol is a bacteriostatic drug that affects cell wall integrity, but the effects of this drug on bacilli are not fully exploited. OBJECTIVE: Based on the need to better investigate the complex mechanism of action of ethambutol, our study presented the proteome profile of M. tuberculosis after different times of ethambutol exposure, aiming to comprehend the dynamics of bacilli response to its effects. M. tuberculosis was exposed to ½ MIC of ethambutol at 24 and 48 hours. The proteins were identified by MALDI-TOF/TOF. RESULTS: The main protein changes occurred in metabolic proteins as dihydrolipoyl dehydrogenase (Rv0462), glutamine synthetase1 (Rv2220), electron transfer flavoprotein subunit beta (Rv3029c) and adenosylhomocysteinase (Rv3248c). CONCLUSION: Considering the functions of these proteins, our results support that the intermediary metabolism and respiration were affected by ethambutol and this disturbance provided proteins that could be explored as additional targets for this drug.

Predicting the Outcomes of New Short-Course Regimens for Multidrug-Resistant Tuberculosis Using Intrahost and Pharmacokinetic-Pharmacodynamic Modeling.

Short-course regimens for multidrug-resistant tuberculosis (MDR-TB) are urgently needed. Limited data suggest that the new drug bedaquiline (BDQ) may have the potential to shorten MDR-TB treatment to less than 6 months when used in conjunction with standard anti-TB drugs. However, the feasibility of BDQ in shortening MDR-TB treatment duration remains to be established. Mathematical modeling provides a platform to investigate different treatment regimens and predict their efficacy. We developed a mathematical model to capture the immune response to TB inside a human host environment. This model was then combined with a pharmacokinetic-pharmacodynamic model to simulate various short-course BDQ-containing regimens. Our modeling suggests that BDQ could reduce MDR-TB treatment duration to just 18 weeks (4 months) while still maintaining a very high treatment success rate (100% for daily BDQ for 2 weeks, or 95% for daily BDQ for 1 week during the intensive phase). The estimated time to bacterial clearance of these regimens ranges from 27 to 33 days. Our findings provide the justification for empirical evaluation of short-course BDQ-containing regimens. If short-course BDQ-containing regimens are found to improve outcomes, then we anticipate clear cost savings and a subsequent improvement in the efficiency of national TB programs.

Optimized Background Regimen for Treatment of Active Tuberculosis with the Next-Generation Benzothiazinone Macozinone (PBTZ169).

The efficacy of the standardized four-drug regimen (comprising isoniazid, rifampin, pyrazinamide, and ethambutol) for the treatment of tuberculosis (TB) is menaced by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis Intensive efforts have been made to develop new antibiotics or to repurpose old drugs, and several of these are currently being evaluated in clinical trials for their antitubercular activity. Among the new candidate drugs is macozinone (MCZ), the piperazine-containing benzothiazinone PBTZ169, which is currently being evaluated in phase I/II clinical trials. Here, we determined the in vitro and in vivo activity of MCZ in combination with a range of anti-TB drugs in order to design a new regimen against active TB. Two-drug combinations with MCZ were tested against M. tuberculosis using checkerboard and CFU enumeration after drug exposure assays. MCZ was observed to have no interactions with all first- and second-line anti-TB drugs. At the MIC of each drug, MCZ with either bedaquiline (BDQ), clofazimine (CLO), delamanid (DMD), or sutezolid (STZ) reduced the bacterial burden by 2 logs compared to that achieved with the drugs alone, indicating synergism. MCZ also displayed synergism with clomiphene (CLM), a potential inhibitor of the undecaprenyl pyrophosphate synthase (UppS) in mycobacteria. For all the other drugs tested in combination with MCZ, no synergistic activity was observed. Neither antagonism nor increased cytotoxicity was found for most combinations, suggesting that MCZ could be added to different TB treatment regimens without any significant adverse effects.

Characterizing the Effects of Glutathione as an Immunoadjuvant in the Treatment of Tuberculosis.

Mycobacterium tuberculosis is the etiological agent that is responsible for causing tuberculosis (TB), which continues to affect millions of people worldwide, and the rate of resistance of M. tuberculosis to antibiotics is ever increasing. We tested the synergistic effects of N-acetyl cysteine (NAC; the precursor molecule for the synthesis of glutathione [GSH]) and individual first-line antibiotics typically given for the treatment of TB, such as isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), to improve the ability of macrophages to control intracellular M. tuberculosis infection. GSH, a pleiotropic antioxidant molecule, has previously been shown to display both antimycobacterial and immune-enhancing effects. Our results indicate that there was not only an increase in beneficial immunomodulatory effects but also a greater reduction in the intracellular viability of M. tuberculosis when macrophages were treated with the combination of antibiotics (INH, RIF, EMB, or PZA) and NAC.

Synergy between Circular Bacteriocin AS-48 and Ethambutol against Mycobacterium tuberculosis.

The increasing incidence of multidrug-resistant Mycobacterium tuberculosis strains and the very few drugs available for treatment are promoting the discovery and development of new molecules that could help in the control of this disease. Bacteriocin AS-48 is an antibacterial peptide produced by Enterococcus faecalis and is active against several Gram-positive bacteria. We have found that AS-48 was active against Mycobacterium tuberculosis, including H37Rv and other reference and clinical strains, and also against some nontuberculous clinical mycobacterial species. The combination of AS-48 with either lysozyme or ethambutol (commonly used in the treatment of drug-susceptible tuberculosis) increased the antituberculosis action of AS-48, showing a synergic interaction. Under these conditions, AS-48 exhibits a MIC close to some MICs of the first-line antituberculosis agents. The inhibitory activity of AS-48 and its synergistic combination with ethambutol were also observed on M. tuberculosis-infected macrophages. Finally, AS-48 did not show any cytotoxicity against THP-1, MHS, and J774.2 macrophage cell lines at concentrations close to its MIC. In summary, bacteriocin AS-48 has interesting antimycobacterial activity in vitro and low cytotoxicity, so further studies in vivo will contribute to its development as a potential additional drug for antituberculosis therapy.

Intact Cell Lipidomics Reveal Changes to the Ratio of Cardiolipins to Phosphatidylinositols in Response to Kanamycin in HeLa and Primary Cells.

Antimicrobial resistance is a major threat the world is currently facing. Development of new antibiotics and the assessment of their toxicity represent important challenges. Current methods for addressing antibiotic toxicity rely on measuring mitochondrial damage using ATP and/or membrane potential as a readout. In this study, we propose an alternative readout looking at changes in the lipidome on intact and unprocessed cells by matrix-assisted laser desorption ionization mass spectrometry. As a proof of principle, we evaluated the impact of known antibiotics (levofloxacin, ethambutol, and kanamycin) on the lipidome of HeLa cells and mouse bone marrow-derived macrophages. Our methodology revealed that clinically relevant concentrations of kanamycin alter the ratio of cardiolipins to phosphatidylinositols. Unexpectedly, only kanamycin had this effect even though all antibiotics used in this study led to a decrease in the maximal mitochondrial respiratory capacity. Altogether, we report that intact cell-targeted lipidomics can be used as a qualitative method to rapidly assess the toxicity of aminoglycosides in HeLa and primary cells. Moreover, these results demonstrate there is no direct correlation between the ratio of cardiolipins to phosphatidylinositols and the maximal mitochondrial respiratory capacity.

Accelerating Early Antituberculosis Drug Discovery by Creating Mycobacterial Indicator Strains That Predict Mode of Action.

Due to the rise of drug-resistant forms of tuberculosis, there is an urgent need for novel antibiotics to effectively combat these cases and shorten treatment regimens. Recently, drug screens using whole-cell analyses have been shown to be successful. However, current high-throughput screens focus mostly on stricto sensu life/death screening that give little qualitative information. In doing so, promising compound scaffolds or nonoptimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early tuberculosis (TB) drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to subinhibitory concentrations of antibiotics with known targets, ciprofloxacin, ethambutol, isoniazid, streptomycin, and rifampin. The resulting data set comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint. Notably, this fingerprint was more distinctive in M. marinum We decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed to respond to DNA damage, cell wall damage, and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so, we identified the putative modes of action for three novel compounds, which confirms the utility of our approach.

Identification of protective postexposure mycobacterial vaccine antigens using an immunosuppression-based reactivation model in the zebrafish.

Roughly one third of the human population carries a latent Mycobacterium tuberculosis infection, with a 5-10% lifetime risk of reactivation to active tuberculosis and further spreading the disease. The mechanisms leading to the reactivation of a latent Mycobacterium tuberculosis infection are insufficiently understood. Here, we used a natural fish pathogen, Mycobacterium marinum, to model the reactivation of a mycobacterial infection in the adult zebrafish (Danio rerio). A low-dose intraperitoneal injection (∼40 colony-forming units) led to a latent infection, with mycobacteria found in well-organized granulomas surrounded by a thick layer of fibrous tissue. A latent infection could be reactivated by oral dexamethasone treatment, which led to disruption of the granuloma structures and dissemination of bacteria. This was associated with the depletion of lymphocytes, especially CD4+ T cells. Using this model, we verified that ethambutol is effective against an active disease but not a latent infection. In addition, we screened 15 mycobacterial antigens as postexposure DNA vaccines, of which RpfB and MMAR_4207 reduced bacterial burdens upon reactivation, as did the Ag85-ESAT-6 combination. In conclusion, the adult zebrafish-M. marinum infection model provides a feasible tool for examining the mechanisms of reactivation in mycobacterial infections, and for screening vaccine and drug candidates.This article has an associated First Person interview with the first author of the paper.

Chemical Genetic Interaction Profiling Reveals Determinants of Intrinsic Antibiotic Resistance in Mycobacterium tuberculosis.

Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in Mycobacterium tuberculosis, we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the M. tuberculosis cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.

In vitro activity of alpha-viniferin isolated from the roots of Carex humilis against Mycobacterium tuberculosis.

This study explores the antitubercular activity of α-viniferin, a bioactive phytochemical compound obtained from Carex humilis. α-Viniferin was active against both drug-susceptible and -resistant strains of Mycobacterium tuberculosis at MIC50s of 4.6 µM in culture broth medium and MIC50s of 2.3-4.6 µM inside macrophages and pneumocytes. In combination with streptomycin and ethambutol, α-viniferin exhibited an additive effect and partial synergy, respectively, against M. tuberculosis H37Rv. α-Viniferin also did not show cytotoxicity in any of the cell lines tested up to a concentration of 147 µM, which gives this compound a selectivity index of >32. Moreover, α-viniferin was active against 3 Staphylococcus species, including methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant Staphylococcus epidermidis (MRSE).

Evaluation of the commercial kit SIRE nitratase for detecting resistant mycobacterium tuberculosis in Brazil

Abstract INTRODUCTION: This study aimed to evaluate a new commercial kit, Kit SIRE Nitratase-PlastLabor, for testing the drug susceptibility of clinical Mycobacterium tuberculosis isolates. METHODS: The accuracy of the Kit SIRE Nitratase was evaluated by examining the susceptibility (streptomycin, isoniazid, rifampicin, and ethambutol) of 40 M. tuberculosis isolates, using the proportion method with Lowenstein-Jensen medium or the BACTEC MGIT 960 system. RESULTS: The detection accuracy for streptomycin, isoniazid, rifampicin, and ethambutol was 95%, 97.5%, 100%, and 80%, respectively. CONCLUSIONS: The exceptional accuracy demonstrated by Kit SIRE Nitratase for isoniazid and rifampicin makes the kit an attractive option for screening M. tuberculosis strain resistance.