Mycobacterium abscessus HelR interacts with RNA polymerase to confer intrinsic rifamycin resistance.

Rifampicin (RIF), the frontline drug against M. tuberculosis, is completely ineffective against M. abscessus, partially due to the presence of an ADP-ribosyltransferase (Arr) that inactivates RIF. Using RNA-seq, we show that exposure of M. abscessus to sublethal doses of RIF and Rifabutin (RBT), a close analog of RIF, results in an ∼25-fold upregulation of Mab_helR in laboratory and clinical isolates. An isogenic deletion in Mab_helR results in RIF/RBT hypersensitivity, and overexpression of Mab_helR confers RIF tolerance in M. tuberculosis. We demonstrate an increased HelR-RNAP association in RIF-exposed bacteria and a MabHelR-mediated dissociation of RNAP from stalled initiation complexes in vitro. Finally, we show that the tip of the PCh-loop of Mab_helR, present in proximity to RIF, is critical for conferring RIF resistance but dispensable for dissociation of stalled RNAP complexes, suggesting that HelR-mediated RIF resistance requires a step in addition to displacement of RIF-stalled RNAP.

HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics.

Rifamycin antibiotics such as rifampin are potent inhibitors of prokaryotic RNA polymerase (RNAP) used to treat tuberculosis and other bacterial infections. Although resistance arises in the clinic principally through mutations in RNAP, many bacteria possess highly specific enzyme-mediated resistance mechanisms that modify and inactivate rifamycins. The expression of these enzymes is controlled by a 19-bp cis-acting rifamycin-associated element (RAE). Guided by the presence of RAE sequences, we identify a helicase-like protein, HelR, in Streptomyces venezuelae that confers broad-spectrum rifamycin resistance. We show that HelR also promotes tolerance to rifamycins, enabling bacterial evasion of the toxic properties of these antibiotics. HelR forms a complex with RNAP and rescues transcription inhibition by displacing rifamycins from RNAP, thereby providing resistance by target protection . Furthermore, HelRs are broadly distributed in Actinobacteria, including several opportunistic Mycobacterial pathogens, offering yet another challenge for developing new rifamycin antibiotics.

Rifampicin drug resistance and host immunity in tuberculosis: more than meets the eye.

Tuberculosis (TB) is the leading cause of death due to an infectious agent, with more than 1.5 million deaths attributed to TB annually worldwide. The global dissemination of drug resistance across Mycobacterium tuberculosis (Mtb) strains, causative of TB, resulted in an estimated 450 000 cases of drug-resistant (DR) TB in 2021. Dysregulated immune responses have been observed in patients with multidrug resistant (MDR) TB, but the effects of drug resistance acquisition and impact on host immunity remain obscure. In this review, we compile studies that span aspects of altered host-pathogen interactions and highlight research that explores how drug resistance and immunity might intersect. Understanding the immune processes differentially induced during DR TB would aid the development of rational therapeutics and vaccines for patients with MDR TB.

Mode of Action of Kanglemycin A, an Ansamycin Natural Product that Is Active against Rifampicin-Resistant Mycobacterium tuberculosis.

Antibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered-compared with rifampicin-conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance.

The human proton pump inhibitors inhibit Mycobacterium tuberculosis rifampicin efflux and macrophage-induced rifampicin tolerance.

Tuberculosis treatment requires months-long combination chemotherapy with multiple drugs, with shorter treatments leading to relapses. A major impediment to shortening treatment is that Mycobacterium tuberculosis becomes tolerant to the administered drugs, starting early after infection and within days of infecting macrophages. Multiple lines of evidence suggest that macrophage-induced drug tolerance is mediated by mycobacterial drug efflux pumps. Here, using assays to directly measure drug efflux, we find that M. tuberculosis transports the first-line antitubercular drug rifampicin through a proton gradient-dependent mechanism. We show that verapamil, a known efflux pump inhibitor, which inhibits macrophage-induced rifampicin tolerance, also inhibits M.tuberculosis rifampicin efflux. As with macrophage-induced tolerance, the calcium channel-inhibiting property of verapamil is not required for its inhibition of rifampicin efflux. By testing verapamil analogs, we show that verapamil directly inhibits M. tuberculosis drug efflux pumps through its human P-glycoprotein (PGP)-like inhibitory activity. Screening commonly used drugs with incidental PGP inhibitory activity, we find many inhibit rifampicin efflux, including the proton pump inhibitors (PPIs) such as omeprazole. Like verapamil, the PPIs inhibit macrophage-induced rifampicin tolerance as well as intramacrophage growth, which has also been linked to mycobacterial efflux pump activity. Our assays provide a facile screening platform for M. tuberculosis efflux pump inhibitors that inhibit in vivo drug tolerance and growth.

Antibiotic perseverance increases the risk of resistance development.

The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular responses. Using single-cell imaging, we studied the growth response of Escherichia coli to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for three out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the population size and led to an up to 40-fold increase in the frequency of antibiotic resistance mutations in gram-negative and gram-positive species. We conclude that antibiotic perseverance is a common phenomenon that has the potential to impact antibiotic resistance development across pathogenic bacteria.

Limiting etioplast gene expression induces apical hook twisting during skotomorphogenesis of Arabidopsis seedlings.

When covered by a layer of soil, seedling development follows a dark-specific program (skotomorphogenesis). In the dark, seedlings consist of small, non-green cotyledons, a long hypocotyl, and an apical hook to protect meristematic cells. We recently highlighted the role played by mitochondria in the high energy-consuming reprogramming of Arabidopsis skotomorphogenesis. Here, the role played by plastids, another energy-supplying organelle, in skotomorphogenesis is investigated. This study was conducted in dark conditions to exclude light signals so as to better focus on those produced by plastids. It was found that limitation of plastid gene expression (PGE) induced an exaggerated apical hook bending. Inhibition of PGE was obtained at the levels of transcription and translation using the antibiotics rifampicin (RIF) and spectinomycin, respectively, as well as plastid RPOTp RNA polymerase mutants. RIF-treated seedlings also showed expression induction of marker nuclear genes for mitochondrial stress, perturbation of mitochondrial metabolism, increased ROS levels, and an augmented capacity of oxygen consumption by mitochondrial alternative oxidases (AOXs). AOXs act to prevent overreduction of the mitochondrial electron transport chain. Previously, we reported that AOX1A, the main AOX isoform, is a key component in the developmental response to mitochondrial respiration deficiency. In this work, we suggest the involvement of AOX1A in the response to PGE dysfunction and propose the importance of signaling between plastids and mitochondria. Finally, it was found that seedling architecture reprogramming in response to RIF was independent of canonical organelle retrograde pathways and the ethylene signaling pathway.

Twice-Daily Dosing of Dolutegravir in Infants on Rifampicin Treatment: A Pharmacokinetic Substudy of the EMPIRICAL Trial.

BACKGROUND: We evaluated dolutegravir pharmacokinetics in infants with human immunodeficiency virus (HIV) receiving dolutegravir twice daily (BID) with rifampicin-based tuberculosis (TB) treatment compared with once daily (OD) without rifampicin. METHODS: Infants with HIV aged 1-12 months, weighing ≥3 kg, and receiving dolutegravir BID with rifampicin or OD without rifampicin were eligible. Six blood samples were taken over 12 (BID) or 24 hours (OD). Dolutegravir pharmacokinetic parameters, HIV viral load (VL) data, and adverse events (AEs) were reported. RESULTS: Twenty-seven of 30 enrolled infants had evaluable pharmacokinetic curves. The median (interquartile range) age was 7.1 months (6.1-9.9), weight was 6.3 kg (5.6-7.2), 21 (78%) received rifampicin, and 11 (41%) were female. Geometric mean ratios comparing dolutegravir BID with rifampicin versus OD without rifampicin were area under curve (AUC)0-24h 0.91 (95% confidence interval, .59-1.42), Ctrough 0.95 (0.57-1.59), Cmax 0.87 (0.57-1.33). One infant (5%) receiving rifampicin versus none without rifampicin had dolutegravir Ctrough <0.32 mg/L, and none had Ctrough <0.064 mg/L. The dolutegravir metabolic ratio (dolutegravir-glucuronide AUC/dolutegravir AUC) was 2.3-fold higher in combination with rifampicin versus without rifampicin. Five of 82 reported AEs were possibly related to rifampicin or dolutegravir and resolved without treatment discontinuation. Upon TB treatment completion, HIV viral load was <1000 copies/mL in 76% and 100% of infants and undetectable in 35% and 20% of infants with and without rifampicin, respectively. CONCLUSIONS: Dolutegravir BID in infants receiving rifampicin resulted in adequate dolutegravir exposure, supporting this treatment approach for infants with HIV-TB coinfection.

Rifampin Based Therapy for Patients With Staphylococcus aureus Native Vertebral Osteomyelitis: A Systematic Review and Meta-analysis.

BACKGROUND: Native vertebral osteomyelitis (NVO) caused by Staphylococcus aureus is associated with high risk of treatment failure and increased morbidity. The role of rifampin-based therapy for the treatment of this condition is controversial. The goal of this systematic review and meta-analysis is to explore the efficacy and safety of rifampin-based therapy for the treatment of S. aureus NVO. METHODS: We searched Cochrane, Embase, Medline, Scopus, and Web of Science databases for studies published up to May 2023, focusing on adults with NVO treated with or without rifampin-containing regimens. A random-effects model meta-analysis estimated relative risks and risk difference with 95% confidence intervals (CI). RESULTS: Thirteen studies (2 randomized controlled trials and 11 comparative cohort studies), comprising 244 patients with S. aureus NVO who received rifampin and 435 who did not, were analyzed. Meta-analysis showed that rifampin-based regimens were associated with lower risk of clinical failure (risk difference, -14%; 95% CI, -19% to -8%; P < .001; I2 = 0%; relative risk, 0.58; 95% CI, .37-.92, P = .02, I2 = 21%). Only 1 study reported on adverse events. All studies had a high or uncertain risk of bias, and the certainty of evidence was rated as very low. CONCLUSIONS: Adjunctive rifampin therapy might be associated with lower risk of S. aureus NVO treatment failure; however, the low certainty of evidence precludes drawing definitive conclusions that would alter clinical practice. A randomized trial is necessary to corroborate these findings.

Pharmacokinetic-Pharmacodynamic Evidence From a Phase 3 Trial to Support Flat-Dosing of Rifampicin for Tuberculosis.

BACKGROUND: The optimal dosing strategy for rifampicin in treating drug-susceptible tuberculosis (TB) is still highly debated. In the phase 3 clinical trial Study 31/ACTG 5349 (NCT02410772), all participants in the control regimen arm received 600 mg rifampicin daily as a flat dose. Here, we evaluated relationships between rifampicin exposure and efficacy and safety outcomes. METHODS: We analyzed rifampicin concentration time profiles using population nonlinear mixed-effects models. We compared simulated rifampicin exposure from flat- and weight-banded dosing. We evaluated the effect of rifampicin exposure on stable culture conversion at 6 months; TB-related unfavorable outcomes at 9, 12, and 18 months using Cox proportional hazard models; and all trial-defined safety outcomes using logistic regression. RESULTS: Our model-derived rifampicin exposure ranged from 4.57 mg · h/L to 140.0 mg · h/L with a median of 41.8 mg · h/L. Pharmacokinetic simulations demonstrated that flat-dosed rifampicin provided exposure coverage similar to the weight-banded dose. Exposure-efficacy analysis (n = 680) showed that participants with rifampicin exposure below the median experienced similar hazards of stable culture conversion and TB-related unfavorable outcomes compared with those with exposure above the median. Exposure-safety analysis (n = 722) showed that increased rifampicin exposure was not associated with increased grade 3 or higher adverse events or serious adverse events. CONCLUSIONS: Flat-dosing of rifampicin at 600 mg daily may be a reasonable alternative to the incumbent weight-banded dosing strategy for the standard-of-care 6-month regimen. Future research should assess the optimal dosing strategy for rifampicin, at doses higher than the current recommendation.

Long-Term Protective Effect of Tuberculosis Preventive Therapy in a Medium/High Tuberculosis Incidence Setting.

BACKGROUND: The duration of the protective effect of tuberculosis preventive therapy (TPT) is controversial. Some studies have found that the protective effect of TPT is lost after cessation of therapy among people with human immunodeficiency virus (HIV) in settings with very high tuberculosis incidence, but others have found long-term protection in low-incidence settings. METHODS: We estimated the incidence rate (IR) of new tuberculosis disease for up to 12 years after randomization to 4 months of rifampin or 9 months of isoniazid, among 991 Brazilian participants in a TPT trial in the state of Rio de Janeiro, with an incidence of 68.6/100 000 population in 2022. The adjusted hazard ratios (aHRs) of independent variables for incident tuberculosis were calculated. RESULTS: The overall tuberculosis IR was 1.7 (95% confidence interval [CI], 1.01- 2.7) per 1000 person-years (PY). The tuberculosis IR was higher among those who did not complete TPT than in those who did (2.9 [95% CI, 1.3-5.6] vs 1.1 [.4-2.3] per 1000 PY; IR ratio, 2.7 [1.0-7.2]). The tuberculosis IR was higher within 28 months after randomization (IR, 3.5 [95% CI, 1.6-6.6] vs 1.1 [.5-2.1] per 1000 PY between 28 and 143 months; IR ratio, 3.1 [1.2-8.2]). Treatment noncompletion was the only variable associated with incident tuberculosis (aHR, 3.2 [95% CI, 1.1-9.7]). CONCLUSIONS: In a mostly HIV-noninfected population, a complete course of TPT conferred long-term protection against tuberculosis.

Environmental and genetic influence on the rate and spectrum of spontaneous mutations in Escherichia coli.

Spontaneous mutations are the ultimate source of novel genetic variation on which evolution operates. Although mutation rate is often discussed as a single parameter in evolution, it comprises multiple distinct types of changes at the level of DNA. Moreover, the rates of these distinct changes can be independently influenced by genomic background and environmental conditions. Using fluctuation tests, we characterized the spectrum of spontaneous mutations in Escherichia coli grown in low and high glucose environments. These conditions are known to affect the rate of spontaneous mutation in wild-type MG1655, but not in a ΔluxS deletant strain - a gene with roles in both quorum sensing and the recycling of methylation products used in E. coli's DNA repair process. We find an increase in AT>GC transitions in the low glucose environment, suggesting that processes relating to the production or repair of this mutation could drive the response of overall mutation rate to glucose concentration. Interestingly, this increase in AT>GC transitions is maintained by the glucose non-responsive ΔluxS deletant. Instead, an elevated rate of GC>TA transversions, more common in a high glucose environment, leads to a net non-responsiveness of overall mutation rate for this strain. Our results show how relatively subtle changes, such as the concentration of a carbon substrate or loss of a regulatory gene, can substantially influence the amount and nature of genetic variation available to selection.

Efficacy of dolutegravir + lamivudine q.d. with food in people with TB/HIV using a rifampicin-based regimen: A retrospective observational case series.

OBJECTIVES: Dolutegravir + lamivudine (DTG + 3TC) is a first-line regimen for people with HIV. However, there are still concerns about its efficacy in people with tuberculosis (TB)/HIV due to the lack of available evidence and drug-drug interaction with rifampicin. METHODS: A single-centre retrospective observational case series was conducted in Guangxi Zhuang Autonomous Region, China. We included all people with TB/HIV on combined use of once-daily (q.d.) dosing DTG + 3TC and rifampicin (RIF)-containing anti-TB regimens between 2020 and 2022. HIV-RNA, CD4 cell counts were collected and analysed. RESULTS: In all, 21 people with HIV (PWH) were included in this study. All the PWH were treatment-naïve and told to take DTG + 3TC q.d. with food. The median age was 53 years, and 71.43% were male. A total of 71.43% PWH had baseline viral load (VL) > 100 000 copies/mL, and 33.33% had baseline VL greater than 500 000 copies/mL. Only one PWH had CD4 cell count greater than 200 cells/µL, and the median CD4 count was 20 cells/µL. A total of 16 PWH started DTG + 3TC after initiation of the RIF-based anti-TB regimen, and the other five PWH initiated DTG + 3TC before the treatment of TB. All the PWH had at least 24 weeks of follow-up visits and all of the TB treatments were successful. A total of 20 PWH (95.24%) achieved viral suppression (VL <50 copies/mL). All detected viral loads between weeks 24 and 48 were less than 200 copies/mL. Among the PWH who started DTG + 3TC after the initiation of RIF-based anti-TB regimen, all achieved viral suppression by week 24 except the non-suppressed PWH. CD4 counts were greatly improved after antiretroviral treatment: the median CD4 counts were raised from 20 to 171 cells/µL at week 48. No serious adverse events were reported. CONCLUSIONS: This case series preliminarily validates the efficacy of DTG + 3TC q.d. with food when combined with RIF-based anti-TB regimens in people with TB/HIV.

Preparation and Characterization of a Rifampicin Coamorphous Material with Tromethamine Coformer: An Experimental-Theoretical Study.

Rifampicin (RIF) is an antibiotic used to treat tuberculosis and leprosy. Even though RIF is a market-available drug, it has a low aqueous solubility, hindering its bioavailability. Among the strategies for bioavailability improvement of poorly soluble drugs, coamorphous systems have been revealed as an alternative in the increase of the aqueous solubility of drug systems and at the same time also increasing the amorphous state stability and dissolution rate when compared with the neat drug. In this work, a new coamorphous form from RIF and tromethamine (TRIS) was synthesized by slow evaporation. Structural, electronic, and thermodynamic properties and solvation effects, as well as drug-coformer intermolecular interactions, were studied through density functional theory (DFT) calculations. Powder X-ray diffraction (PXRD) data allowed us to verify the formation of a new coamorphous. In addition, the DFT study indicates a possible intermolecular interaction by hydrogen bonds between the available amino and carbonyl groups of RIF and the hydroxyl and amino groups of TRIS. The theoretical spectra obtained are in good agreement with the experimental data, suggesting the main interactions occurring in the formation of the coamorphous system. PXRD was used to study the physical stability of the coamorphous system under accelerated ICH conditions (40 °C and 75% RH), indicating that the material remained in an amorphous state up to 180 days. The thermogravimetry result of this material showed a good thermal stability up to 153 °C, and differential scanning calorimetry showed that the glass temperature (Tg) was at 70.0 °C. Solubility studies demonstrated an increase in the solubility of RIF by 5.5-fold when compared with its crystalline counterpart. Therefore, this new material presents critical parameters that can be considered in the development of new coamorphous formulations.

Lowering mortality risk in CR-HvKP infection in intestinal immunohistological and microbiota restoration.

Gut damage during carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HvKP) infection is associated with a death risk. Understanding the mechanisms by which CR-HvKP causes intestinal damage and gut microbiota alteration, and the impact on immunity, is crucial for developing therapeutic strategies. This study investigated if gastrointestinal tract damage and disruption of gut microbiota induced by CR-HvKP infection undermined host immunity and facilitated multi-organ invasion of CR-HvKP; whether the therapeutic value of the rifampicin (RIF) and zidovudine (ZDV) combination was attributed to their ability to repair damages and restore host immunity was determined. A sepsis model was utilized to assess the intestinal pathological changes. Metagenomic analysis was performed to characterize the alteration of gut microbiota. The effects of the RIF and ZDV on suppressing inflammatory responses and improving immune functions and gut microbiota were evaluated by immunopathological and transcriptomic analyses. Rapid colonic damage occurred upon activation of the inflammation signaling pathways during lethal infections. Gut inflammation compromised host innate immunity and led to a significant decrease in probiotics abundance, including Bifidobacterium and Lactobacillus. Treatment with combination drugs significantly attenuated the inflammatory response, up-regulated immune cell differentiation signaling pathways, and promoted the abundance of Bifidobacterium (33.40 %). Consistently, supplementation of Bifidobacterium alone delayed the death in sepsis model. Gut inflammation and disrupted microbiota are key disease features of CR-HvKP infection but can be reversed by the RIF and ZDV drug combination. The finding that these drugs can restore host immunity through multiple mechanisms is novel and deserves further investigation of their clinical application potential.

Effects of rifampicin on the pharmacokinetics and safety of carotegrast methyl in healthy subjects: A randomized 2 × 2 crossover study.

AIMS: To evaluate the effect of the combination of carotegrast methyl with rifampicin, a potent inhibitor of organic anion transporter polypeptide, on the pharmacokinetics (PKs), safety and tolerability of carotegrast methyl. METHODS: In this 2 × 2 crossover study in 20 healthy Japanese adults, 10 subjects received carotegrast methyl 960 mg and rifampicin 600 mg on day 1 and received carotegrast methyl 960 mg on day 8. The subjects in the other sequence received the same treatments but in the opposite order. The 90% confidence interval (CI) of the geometric mean ratio of the Cmax and AUC0-t for carotegrast, the main active metabolite of carotegrast methyl, with/without rifampicin was calculated. If the 90% CI fell within the range of 0.80-1.25, this indicated the absence of any drug-drug interaction. Adverse events (AEs) were monitored. RESULTS: The geometric mean ratios (90% CI) of the Cmax and AUC0-t for carotegrast with/without rifampicin were 4.78 (3.64-6.29) and 5.59 (4.60-6.79), respectively, indicating that carotegrast has a PK interaction with rifampicin. The combination with rifampicin increased the exposure of carotegrast and also that of its metabolites. The incidence of any AEs with/without rifampicin was five (25.0%) and one (5.0%), respectively. CONCLUSIONS: Coadministration of carotegrast methyl with rifampicin significantly increased the exposure of carotegrast compared with carotegrast methyl administration alone. In this single dose study, the incidence of AEs of carotegrast methyl with rifampicin increased compared with carotegrast methyl alone, but the incidence of adverse drug reactions did not increase with combination administration.

Predicting Drug-Drug Interactions Involving Rifampicin Using a Semi-mechanistic Hepatic Compartmental Model.

AIMS: To develop a semi-mechanistic hepatic compartmental model to predict the effects of rifampicin, a known inducer of CYP3A4 enzyme, on the metabolism of five drugs, in the hope of informing dose adjustments to avoid potential drug-drug interactions. METHODS: A search was conducted for DDI studies on the interactions between rifampicin and CYP substrates that met specific criteria, including the availability of plasma concentration-time profiles, physical and absorption parameters, pharmacokinetic parameters, and the use of healthy subjects at therapeutic doses. The semi-mechanistic model utilized in this study was improved from its predecessors, incorporating additional parameters such as population data (specifically for Chinese and Caucasians), virtual individuals, gender distribution, age range, dosing time points, and coefficients of variation. RESULTS: Optimal parameters were identified for our semi-mechanistic model by validating it with clinical data, resulting in a maximum difference of approximately 2-fold between simulated and observed values. PK data of healthy subjects were used for most CYP3A4 substrates, except for gilteritinib, which showed no significant difference between patients and healthy subjects. Dose adjustment of gilteritinib co-administered with rifampicin required a 3-fold increase of the initial dose, while other substrates were further tuned to achieve the desired drug exposure. CONCLUSIONS: The pharmacokinetic parameters AUCR and CmaxR of drugs metabolized by CYP3A4, when influenced by Rifampicin, were predicted by the semi-mechanistic model to be approximately twice the empirically observed values, which suggests that the semi-mechanistic model was able to reasonably simulate the effect. The doses of four drugs adjusted via simulation to reduce rifampicin interaction.

A reconstructed genome-scale metabolic model of Helicobacter pylori for predicting putative drug targets in clarithromycin and rifampicin resistance conditions.

BACKGROUND: Helicobacter pylori is considered a true human pathogen for which rising drug resistance constitutes a drastic concern globally. The present study aimed to reconstruct a genome-scale metabolic model (GSMM) to decipher the metabolic capability of H. pylori strains in response to clarithromycin and rifampicin along with identification of novel drug targets. MATERIALS AND METHODS: The iIT341 model of H. pylori was updated based on genome annotation data, and biochemical knowledge from literature and databases. Context-specific models were generated by integrating the transcriptomic data of clarithromycin and rifampicin resistance into the model. Flux balance analysis was employed for identifying essential genes in each strain, which were further prioritized upon being nonhomologs to humans, virulence factor analysis, druggability, and broad-spectrum analysis. Additionally, metabolic differences between sensitive and resistant strains were also investigated based on flux variability analysis and pathway enrichment analysis of transcriptomic data. RESULTS: The reconstructed GSMM was named as HpM485 model. Pathway enrichment and flux variability analyses demonstrated reduced activity in the ribosomal pathway in both clarithromycin- and rifampicin-resistant strains. Also, a significant decrease was detected in the activity of metabolic pathways of clarithromycin-resistant strain. Moreover, 23 and 16 essential genes were exclusively detected in clarithromycin- and rifampicin-resistant strains, respectively. Based on prioritization analysis, cyclopropane fatty acid synthase and phosphoenolpyruvate synthase were identified as putative drug targets in clarithromycin- and rifampicin-resistant strains, respectively. CONCLUSIONS: We present a robust and reliable metabolic model of H. pylori. This model can predict novel drug targets to combat drug resistance and explore the metabolic capability of H. pylori in various conditions.

Amphiphilic tribasic galactosamines potentiate rifampicin in Gram-negative bacteria at low Mg++/Ca++concentrations.

Many antibiotics specific to Gram-positive bacteria like rifampicin (RIF) are inactive in Gram-negative bacteria because of outer membrane (OM) impermeability. Enhancing the OM permeability of these antibiotics with the help of OM perturbants is a promising strategy to develop new agents against Gram-negative bacteria. Here we report the synthesis and biological properties of amphiphilic tribasic galactosamines as potential RIF potentiators. Our results demonstrate that tribasic galactose-based amphiphiles potentiate RIF in multidrug-resistant Acinetobacter baumannii and Escherichia coli but not Pseudomonas aeruginosa in low salt-containing media. Under these conditions, lead compounds 20, 22 and 35 lowered the minimum inhibitory concentration of RIF by 64- to 256-fold against Gram-negative bacteria. However, the RIF-potentiating effect was reduced when bivalent Mg++ or Ca++ ions were added in the media at physiological concentrations. Overall, our results indicate that amphiphilic tribasic galactosamine-based compounds show reduced RIF-potentiating effects when compared to amphiphilic tobramycin antibiotics at physiological salt concentrations.

Accurate and affordable detection of rifampicin and isoniazid resistance in Tuberculosis sputum specimens by multiplex PCR-multiple probes melting analysis.

BACKGROUND: Escalating cases of multidrug-resistant tuberculosis (MDR-TB) pose a major challenge to global TB control efforts, necessitating innovative diagnostics to empower decentralized detection of gene mutations associated with resistance to rifampicin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis (M. tuberculosis) in resource-constrained settings. METHODS: Combining multiplex fluorescent PCR and Multiple Probes Melting Analysis, we identified mutations in the rpoB, katG, ahpC and inhA genes from sputum specimens. We first constructed a reference plasmid library comprising 40 prevalent mutations in the target genes' resistance determining regions and promoters, serving as positive controls. Our assay utilizes a four-tube asymmetric PCR method with specifically designed molecular beacon probes, enabling simultaneous detection of all 40 mutations. We evaluated the assay's effectiveness using DNA isolated from 50 clinically confirmed M. tuberculosis sputum specimens, comparing our results with those obtained from Sanger sequencing and retrospective validation involving bacteriological culture and phenotypic drug susceptibility testing (pDST). We also included the commercial Xpert MTB/RIF assay for accuracy comparison. RESULTS: Our data demonstrated remarkable sensitivity in detecting resistance to RIF and INH, achieving values of 93.33% and 95.24%, respectively, with a specificity of 100%. The concordance between our assay and pDST was 98.00%. Furthermore, the accuracy of our assay was comparable to both Sanger sequencing and the Xpert assay. Importantly, our assay boasts a 4.2-h turnaround time and costs only $10 per test, making it an optimal choice for peripheral healthcare settings. CONCLUSION: These findings highlight our assay's potential as a promising tool for rapidly, accurately, and affordably detecting MDR-TB.