First detection of a Mycobacterium tuberculosis XDR clinical isolate harbouring an RpoB I491F mutation in a Ukrainian patient treated in Germany, October 2023.

This report documents the case of a Ukrainian patient infected with an extensively drug-resistant (XDR) lineage 2 Mycobacterium tuberculosis strain harbouring the rifampicin resistance mutation RpoB I491F. This mutation is not detected by routine molecular WHO-recommended rapid diagnostics, complicating the detection and treatment of these strains. The occurrence of such mutations underscores the need for enhanced diagnostic techniques and tailored treatment regimens, especially in eastern Europe where lineage 2 strains and XDR-tuberculosis are prevalent.

The mechanism of Gejie Zhilao Pill in treating tuberculosis based on network pharmacology and molecular docking verification.

Introduction: Gejie Zhilao Pill (GJZLP), a traditional Chinese medicine formula is known for its unique therapeutic effects in treating pulmonary tuberculosis. The aim of this study is to further investigate its underlying mechanisms by utilizing network pharmacology and molecular docking techniques. Methods: Using TCMSP database the components, potential targets of GJZLP were identified. Animal-derived components were supplemented through the TCMID and BATMAN-TCM databases. Tuberculosis-related targets were collected from the TTD, OMIM, and GeneCards databases. The intersection target was imported into the String database to build the PPI network. The Metascape platform was employed to carry out Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Heatmaps were generated through an online platform (https://www.bioinformatics.com.cn). Molecular docking was conducted between the core targets and core compounds to explore their binding strengths and patterns at the molecular level. Results: 61 active ingredients and 118 therapeutic targets were identified. Quercetin, Luteolin, epigallocatechin gallate, and beta-sitosterol showed relatively high degrees in the network. IL6, TNF, JUN, TP53, IL1B, STAT3, AKT1, RELA, IFNG, and MAPK3 are important core targets. GO and KEGG revealed that the effects of GJZLP on tuberculosis mainly involve reactions to bacterial molecules, lipopolysaccharides, and cytokine stimulation. Key signaling pathways include TNF, IL-17, Toll-like receptor and C-type lectin receptor signaling. Molecular docking analysis demonstrated a robust binding affinity between the core compounds and the core proteins. Stigmasterol exhibited the lowest binding energy with AKT1, indicating the most stable binding interaction. Discussion: This study has delved into the efficacious components and molecular mechanisms of GJZLP in treating tuberculosis, thereby highlighting its potential as a promising therapeutic candidate for the treatment of tuberculosis.

Epidemiology and antimicrobial resistance of Mycobacterium spp. in the United Arab Emirates: a retrospective analysis of 12 years of national antimicrobial resistance surveillance data.

Introduction: The Eastern Mediterranean Regional Office (EMRO) region accounts for almost 8% of all global Mycobacterium tuberculosis (TB) cases, with TB incidence rates ranging from 1 per 100,000 per year in the United Arab Emirates (UAE) to 204 per 100,000 in Djibouti. The national surveillance data from the Middle East and North Africa (MENA) region on the epidemiology and antimicrobial resistance trends of TB, including MDR-TB remains scarce. Methods: A retrospective 12-year analysis of N = 8,086 non-duplicate diagnostic Mycobacterium tuberculosis complex (MTB complex) isolates from the UAE was conducted. Data were generated through routine patient care during the 2010-2021 years, collected by trained personnel and reported by participating surveillance sites to the UAE National Antimicrobial Resistance (AMR) Surveillance program. Data analysis was conducted with WHONET, a windows-based microbiology laboratory database management software developed by the World Health Organization Collaborating Center for Surveillance of Antimicrobial Resistance, Boston, United States (https://whonet.org/). Results: A total of 8,086 MTB-complex isolates were analyzed. MTB-complex was primarily isolated from respiratory samples (sputum 80.1%, broncho-alveolar lavage 4.6%, pleural fluid 4.1%). Inpatients accounted for 63.2%, including 1.3% from ICU. Nationality was known for 84.3% of patients, including 3.8% Emiratis. Of UAE non-nationals, 80.5% were from 110 countries, most of which were Asian countries. India accounted for 20.8%, Pakistan 13.6%, Philippines 12.7%, and Bangladesh 7.8%. Rifampicin-resistant MTB-complex isolates (RR-TB) were found in 2.8% of the isolates, resistance to isoniazid, streptomycin, pyrazinamide, and ethambutol, was 8.9, 6.9, 3.4 and 0.4%, respectively. A slightly increasing trend of resistance among MTB-complex was observed for rifampicin from 2.5% (2010) to 2.8% (2021). Conclusion: Infections due to MTB-complex are relatively uncommon in the United Arab Emirates compared to other countries in the MENA region. Most TB patients in the UAE are of Asian origin, mainly from countries with a high prevalence of TB. Resistance to first line anti-tuberculous drugs is generally low, however increasing trends for MDR-TB mainly rifampicin linked resistance is a major concern. MDR-TB was not associated with a higher mortality, admission to ICU, or increased length of hospitalization as compared to non-MDR-TB.

The Nitrofuran-Warhead-Equipped Spirocyclic Azetidines Show Excellent Activity against Mycobacterium tuberculosis.

A series of 21 new 7'H-spiro[azetidine-3,5'-furo [3,4-d]pyrimidine]s substituted at the pyrimidine ring second position were synthesized. The compounds showed high antibacterial in vitro activity against M. tuberculosis. Two compounds had lower minimum inhibitory concentrations against Mtb (H37Rv strain) compared with isoniazid. The novel spirocyclic scaffold shows excellent properties for anti-tuberculosis drug development.

Imidazoquinoline Derivatives as Potential Inhibitors of InhA Enzyme and Mycobacterium tuberculosis.

Tuberculosis is a serious public health problem worldwide. The search for new antibiotics has become a priority, especially with the emergence of resistant strains. A new family of imidazoquinoline derivatives, structurally analogous to triazolophthalazines, which had previously shown good antituberculosis activity, were designed to inhibit InhA, an essential enzyme for Mycobacterium tuberculosis survival. Over twenty molecules were synthesized and the results showed modest inhibitory efficacy against the protein. Docking experiments were carried out to show how these molecules could interact with the protein's substrate binding site. Disappointingly, unlike triazolophthlazines, these imidazoquinoline derivatives showed an absence of inhibition on mycobacterial growth.

Inhibition of M. tuberculosis and human ATP synthase by BDQ and TBAJ-587.

Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.

Synthesis and characterisation of antimicrobial metal-organic frameworks as multi-drug carriers.

Antibiotic resistance is a significant global concern, necessitating the development of either new antibiotics or advanced delivery methods. With this in mind, we report on the synthesis and characterisation of a new family of Metal-Organic Frameworks (MOFs), OnG6 MOFs, designed to act as multi-drug carriers for bacterial infection treatment. OnG6 is based on the pro-drug 4,4'-azodisalicylic acid (AZDH4), which in vivo produces two equivalents of para-aminosalicylic acid (ASA), a crucial drug for M. tuberculosis treatment. X-ray and computational studies revealed that OnG6 MOFs are mesoporous MOFs with etb topology and an [M2(AZD)] formula (M = Zn, OnG6-Zn; Mg, OnG6-Mg; Cu, OnG6-Cu; and Co, OnG6-Co), featuring 1-dimensional channel type pores of 25 Å diameter. OnG6 MOFs are the first reported MOFs bearing the ligand AZDH4, joining the family of mesoporous MOFs arranged in a honeycomb pattern. They absorb isoniazid (INH) and ciprofloxacin (CIPRO) with the former being a specific antibiotic for M. tuberculosis, and the latter being a broader-spectrum antibiotic. The stability of the MOFs and their capacity for antibiotic uptake depend on the nature of the metal ion, with OnG6-Mg demonstrating the highest drug absorption. The antimicrobial activity of these species was assessed against S. aureus and E. coli, revealing that the carriers containing CIPRO displayed optimal efficacy.

Association between HIV and acquisition of rifamycin resistance with first-line TB treatment: a systematic review and meta-analysis.

BACKGROUND: Multi-drug or rifamycin-resistant tuberculosis (MDR/RR-TB) is an important public health concern, including in settings with high HIV prevalence. TB drug resistance can be directly transmitted or arise through resistance acquisition during first-line TB treatment. Limited evidence suggests that people living with HIV (PLHIV) might have an increased risk of acquired rifamycin-resistance (ARR). METHODS: To assess HIV as a risk factor for ARR during first-line TB treatment, a systematic review and meta-analysis was conducted. ARR was defined as rifamycin-susceptibility at treatment start with rifamycin-resistance diagnosed during or at the end of treatment, or at recurrence. PubMed/MEDLINE, CINAHL, Cochrane Library, and Google Scholar databases were searched from inception to 23 May 2024 for articles in English; conference abstracts were also searched from 2004 to 2021. The Mantel-Haenszel random-effects model was used to estimate the pooled odds ratio of any association between HIV and ARR among individuals receiving first-line TB treatment. RESULTS: Ten studies that included data collected between 1990 and 2014 were identified: five from the United States, two from South Africa and one each from Uganda, India and Moldova. A total of 97,564 individuals were included across all studies, with 13,359 (13.7%) PLHIV. Overall, 312 (0.32%) acquired rifamycin-resistance, among whom 115 (36.9%) were PLHIV. The weighted odds of ARR were 4.57 (95% CI, 2.01-10.42) times higher among PLHIV compared to HIV-negative individuals receiving first-line TB treatment. CONCLUSION: The available data, suggest that PLHIV have an increased ARR risk during first-line TB treatment. Further research is needed to clarify specific risk factors, including advanced HIV disease and TB disease severity. Given the introduction of shorter, 4-month rifamycin-based regimens, there is an urgent need for additional data on ARR, particularly for PLHIV. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42022327337.

Assembly-Induced Membrane Selectivity of Artificial Model Peptides through Entropy-Enthalpy Competition.

Peptide design and drug development offer a promising solution for combating serious diseases or infections. In this study, using an AI-human negotiation approach, we have designed a class of minimal model peptides against tuberculosis (TB), among which K7W6 exhibits potent efficacy attributed to its assembly-induced function. Comprising lysine and tryptophan with an amphiphilic α-helical structure, the K7W6 sequence exhibits robust activity against various infectious bacteria causing TB (including clinically isolated and drug-resistant strains) both in vitro and in vivo. Moreover, it synergistically enhances the effectiveness of the first-line antibiotic rifampicin while displaying low potential for inducing drug resistance and minimal toxicity toward mammalian cells. Biophysical experiments and simulations elucidate that K7W6's exceptional performance can be ascribed to its highly selective and efficient membrane permeabilization activity induced by its distinctive self-assembly behavior. Additionally, these assemblies regulate the interplay between enthalpy and entropy during K7W6-membrane interaction, leading to the peptide's two-step mechanism of membrane interaction. These findings provide valuable insights into rational design principles for developing advanced peptide-based drugs while uncovering the functional role played by assembly.

Diagnostic accuracy of the Xpert® MTB/XDR assay for detection of Isoniazid and second-line antituberculosis drugs resistance at central TB reference laboratory in Tanzania.

INTRODUCTION: Early diagnosis of tuberculosis (TB) and universal access to drug-susceptibility testing (DST) are critical elements of the WHO End TB Strategy. Current rapid tests (e.g., Xpert® MTB/RIF and Ultra-assays) can detect rifampicin resistance-conferring mutations, but cannot detect resistance to Isoniazid and second-line anti-TB agents. Although Line Probe Assay is capable of detecting resistance to second-line anti-TB agents, it requires sophisticated laboratory infrastructure and advanced skills which are often not readily available in settings replete with TB. A rapid test capable of detecting Isoniazid and second-line anti-TB drug resistance is highly needed. METHODS: We conducted a diagnostic accuracy study to evaluate a new automated Xpert MTB/XDR 10-colour assay for rapid detection of Isoniazid and second-line drugs, including ethionamide, fluoroquinolones, and injectable drugs (Amikacin, Kanamycin, and Capreomycin). Positive Xpert MTB/RIF respiratory specimens were prospectively collected through routine diagnosis and surveillance of drug resistance at the Central TB Reference Laboratory in Tanzania. Specimens were tested by both Xpert XDR assay and LPA against culture-based phenotypic DST as the reference standard. FINDINGS: We analysed specimens from 151 TB patients with a mean age (SD) of 36.2 (12.7) years. The majority (n = 109, 72.2%) were males. The sensitivity for Xpert MTB/XDR was 93.5% (95% CI, 87.4-96.7); for Isoniazid, 96.6 (95% CI, 92.1-98.6); for Fluoroquinolone, 98.7% (95% Cl 94.8-99.7); for Amikacin, 96.6%; and (95% CI 92.1-98.6) for Ethionamide. Ethionamide had the lowest specificity of 50% and the highest was 100% for Fluoroquinolone. The diagnostic performance was generally comparable to that of LPA with slight variations between the two assays. The non-determinate rate (i.e., invalid M. tuberculosis complex detection) of Xpert MTB/XDR was 2·96%. CONCLUSION: The Xpert MTB/XDR demonstrated high sensitivity and specificity for detecting resistance to Isoniazid, Fluoroquinolones, and injectable agents. This assay can be used in clinical settings to facilitate rapid diagnosis of mono-isoniazid and extensively drug-resistant TB.

Rapid Gene Silencing Followed by Antimicrobial Susceptibility Testing for Target Validation in Antibiotic Discovery.

Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.

A Microtiter Plate Assay at Acidic pH to Identify Potentiators that Enhance Pyrazinamide Activity Against Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a key component of chemotherapy for the treatment of drug-susceptible tuberculosis (TB) and is likely to continue to be included in new drug combinations. Potentiation of PZA could be used to reduce the emergence of resistance, shorten treatment times, and lead to a reduction in the quantity of PZA consumed by patients, thereby reducing the toxic effects. Acidified medium is required for the activity of PZA against Mycobacterium tuberculosis. In vitro assessments of pyrazinamide activity are often avoided because of the lack of standardization, which has led to a lack of effective in vitro tools for assessing and/or enhancing PZA activity.We have developed and optimized a novel, robust, and reproducible, microtiter plate assay, that centers around acidity levels that are low enough for PZA activity. The assay can be applied to the evaluation of novel compounds for the identification of potentiators that enhance PZA activity. In this assay, potentiation of PZA is demonstrated to be statistically significant with the addition of rifampicin (RIF), which can, therefore, be used as a positive control. Conversely, norfloxacin demonstrates no potentiating activity with PZA and can be used as a negative control. The method, and the associated considerations, described here, can be adapted in the search for potentiators of other antimicrobials.

Phylogenetic Survival Analysis.

Going back in time through a phylogenetic tree makes it possible to evaluate ancestral genomes and assess their potential to acquire key polymorphisms of interest over evolutionary time. Knowledge of this kind may allow for the emergence of key traits to be predicted and pre-empted from currently circulating strains in the future. Here, we present a novel genome-wide survival analysis and use the emergence of drug resistance in Mycobacterium tuberculosis as an example to demonstrate the potential and utility of the technique.

Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing: From Sequence Data to Resistance Profiles.

Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many first- and second-line anti-tuberculosis drugs. However, bioinformatic pipelines and mutation catalogs to predict antimicrobial resistances in MTBC isolates are often customized and detailed protocols are difficult to access. Here, we provide a step-by-step workflow for the processing and interpretation of short-read sequencing data and give an overview of available analysis pipelines.

Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing.

Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in many countries. WGS protocols usually start with the preparation of a DNA-library: the critical first step in the process. A DNA-library represents the genomic content of a DNA sample and consists of unique short DNA fragments. Although available DNA-library protocols come with manufacturer instructions, details of the entire process, including quality controls, instrument parameters, and run evaluations, often need to be developed and customized by each laboratory to implement WGS technology effectively. Here, we provide a detailed workflow for a DNA-library preparation based on an adapted Illumina protocol optimized for the reduction of reagent costs.

Bedaquiline: An Insight Into its Clinical Use in Multidrug-Resistant Pulmonary Tuberculosis.

Every year, the World Health Organization reports 500,000 new cases of drug-resistant tuberculosis (TB), which poses a serious global danger. The increased number of XDR-TB and MDR-TB cases reported worldwide necessitates the use of new therapeutic approaches. The main issues with the antitubercular medications now in use for the treatment of multidrug-resistant tuberculosis are their poor side effect profile, reduced efficacy, and antimicrobial resistance. One possible remedy for these problems is bedaquiline. The need for better treatment strategies is highlighted by the strong minimum inhibitory concentrations that bedaquiline (BDQ), a novel anti-TB medicine, exhibits against both drug-resistant and drug-susceptible TB. Bedaquiline may be able to help with these problems. Bedaquiline is a medication that is first in its class and has a distinct and particular mode of action. Bedaquiline is an ATP synthase inhibitor that is specifically directed against Mycobacterium tuberculosis and some nontuberculous mycobacteria. It is metabolized by CYP3A4. Bedaquiline preclinical investigations revealed intralesional drug biodistribution. The precise intralesional and multi-compartment pharmacokinetics of bedaquiline were obtained using PET bioimaging and high-resolution autoradiography investigations. Reduced CFU counts were observed in another investigation after a 12-week course of therapy. Meta-analyses and systematic reviews of phase II trials on bedaquiline's efficacy in treating drug-resistant tuberculosis in patients reported higher rates of cure, better culture conversion, and lower death rates when taken in conjunction with a background regimen. Here is a thorough medication profile for bedaquiline to aid medical professionals in treating individuals with tuberculosis.

Apramycin has high in vitro activity against Mycobacterium tuberculosis.

Introduction. Aminoglycoside antibiotics such as amikacin and kanamycin are important components in the treatment of Mycobacterium tuberculosis (Mtb) infection. However, more and more clinical strains are found to be aminoglycoside antibiotic-resistant. Apramycin is another kind of aminoglycoside antibiotic that is commonly used to treat infections in animals.Hypothesis. Apramycin may have in vitro activity against Mtb.Aim. This study aims to evaluate the efficacy of apramycin against Mtb in vitro and determine its epidemiological cut-off (ECOFF) value.Methodology. One hundred Mtb isolates, including 17 pansusceptible and 83 drug-resistant tuberculosis (DR-TB) strains, were analysed for apramycin resistance using the MIC assay.Results. Apramycin exhibited significant inhibitory activity against Mtb clinical isolates, with an MIC50 of 0.5 µg ml-1 and an MIC90 of 1 µg ml-1. We determined the tentative ECOFF value as 1 µg ml-1 for apramycin. The resistant rates of multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant (pre-XDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains were 12.12 % (4/33), 20.69 % (6/29) and 66.67 % (14/21), respectively. The rrs gene A1401G is associated with apramycin resistance, as well as the cross-resistance between apramycin and other aminoglycosides.Conclusion. Apramycin shows high in vitro activity against the Mtb clinical isolates, especially the MDR-TB clinical isolates. This encouraging discovery calls for more research on the functions of apramycin in vivo and as a possible antibiotic for the treatment of drug-resistant TB.