The importance of heteroresistance and efflux pumps in bedaquiline-resistant Mycobacterium tuberculosis isolates from Iran.

BACKGROUND: Tuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates. METHODS: Nineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP). RESULTS: All 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ. CONCLUSIONS: Our results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.

Assessment for Antibodies to Rifapentine and Isoniazid in Persons Developing Flu-like Reactions During Treatment of Latent Tuberculosis Infection.

BACKGROUND: Flu-like reactions can occur after exposure to rifampin, rifapentine, or isoniazid. Prior studies have reported the presence of antibodies to rifampin, but associations with underlying pathogenesis are unclear. METHODS: We evaluated PREVENT TB study participants who received weekly isoniazid + rifapentine for 3 months (3HP) or daily isoniazid for 9 months (9H) as treatment for M. tuberculosis infection. Flu-like reaction was defined as a grade ≥2 of any of flu-like symptoms. Controls (3HP or 9H) did not report flu-like reactions. We developed a competitive enzyme-linked immunosorbent assays (ELISA) to detect antibodies against rifapentine, isoniazid, rifampin, and rifapentine metabolite. RESULTS: Among 128 participants, 69 received 3HP (22 with flu-like reactions; 47 controls) and 59 received 9H (12 with flu-like reactions; 47 controls). In participants receiving 3HP, anti-rifapentine IgG was identified in 2/22 (9%) participants with flu-like reactions and 6/47 (13%) controls (P = 0.7), anti-isoniazid IgG in 2/22 (9%) participants with flu-like reactions and 4/47 (9%) controls (P = 0.9), and anti-rifapentine metabolite IgG in 2/47 (4%) controls (P = 0.9). Among participants receiving 9H, IgG and IgM anti-isoniazid antibodies were each present in 4/47 (9%) controls, respectively, but none among participants with flu-like reactions; anti-rifapentine IgG antibodies were not present in any participants with flu-like reactions or controls. CONCLUSIONS: We detected anti-rifapentine, anti-isoniazid, and anti-rifapentine metabolite antibodies, but the proportions of participants with antibodies were low, and did not differ between participants with flu-like reactions and those without such reactions. This suggests that flu-like reactions associated with 3HP and 9H were not antibody-mediated.

Design, synthesis and characterization of ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate as anti-tubercular agents: In silico screening for possible target identification.

A thorough search for the development of innovative drugs to treat tuberculosis, especially considering the urgent need to address developing drug resistance, we report here a synthetic series of ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o) as potent anti-tubercular agents. These morpholino-indolizines were synthesized by reacting 4-morpholino pyridinium salts, with various electron-deficient acetylenes to afford the ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o). All synthesized intermediate and final compounds are characterized by spectroscopic methods such as 1H NMR, 13C NMR and HRMS and further examined for their anti-tubercular activity against the M. tuberculosis H37Rv strain (ATCC 27294-American type cell culture). All the compounds screened for anti-tubercular activity in the range of 6.25-50 µM against the H37Rv strain of Mycobacterium tuberculosis. Compound 5g showed prominent activity with MIC99 2.55 µg/mL whereas compounds 5d and 5j showed activity with MIC99 18.91 µg/mL and 25.07 µg/mL, respectively. In silico analysis of these compounds revealed drug-likeness. Additionally, the molecular target identification for Malate synthase (PDB 5CBB) is attained by computational approach. The compound 5g with a MIC99 value of 2.55 µg/mL against M. tuberculosis H37Rv emerged as the most promising anti-TB drug and in silico investigations suggest Malate synthase (5CBB) might be the compound's possible target.

Detection of Mtb and NTM: preclinical validation of a new asymmetric PCR-binary deoxyribozyme sensor assay.

Tuberculosis (TB) and infectious diseases caused by non-tuberculous mycobacteria (NTM) are global concerns. The development of a rapid and accurate diagnostic method, capable of detecting and identifying different mycobacteria species, is crucial. We propose a molecular approach, the BiDz-TB/NTM, based on the use of binary deoxyribozyme (BiDz) sensors for the detection of Mycobacterium tuberculosis (Mtb) and NTM of clinical interest. A panel of DNA samples was used to evaluate Mtb-BiDz, Mycobacterium abscessus/Mycobacterium chelonae-BiDz, Mycobacterium avium-BiDz, Mycobacterium intracellulare/Mycobacterium chimaera-BiDz, and Mycobacterium kansasii-BiDz sensors in terms of specificity, sensitivity, accuracy, and limit of detection. The BiDz sensors were designed to hybridize specifically with the genetic signatures of the target species. To obtain the BiDz sensor targets, amplification of a fragment containing the hypervariable region 2 of the 16S rRNA was performed, under asymmetric PCR conditions using the reverse primer designed based on linear-after-the-exponential principles. The BiDz-TB/NTM was able to correctly identify 99.6% of the samples, with 100% sensitivity and 0.99 accuracy. The individual values of specificity, sensitivity, and accuracy, obtained for each BiDz sensor, satisfied the recommendations for new diagnostic methods, with sensitivity of 100%, specificity and accuracy ranging from 98% to 100% and from 0.98 to 1.0, respectively. The limit of detection of BiDz sensors ranged from 12 genome copies (Mtb-BiDz) to 2,110 genome copies (Mkan-BiDz). The BiDz-TB/NTM platform would be able to generate results rapidly, allowing the implementation of the appropriate therapeutic regimen and, consequently, the reduction of morbidity and mortality of patients.IMPORTANCEThis article describes the development and evaluation of a new molecular platform for accurate, sensitive, and specific detection and identification of Mycobacterium tuberculosis and other mycobacteria of clinical importance. Based on BiDz sensor technology, this assay prototype is amenable to implementation at the point of care. Our data demonstrate the feasibility of combining the species specificity of BiDz sensors with the sensitivity afforded by asymmetric PCR amplification of target sequences. Preclinical validation of this assay on a large panel of clinical samples supports the further development of this diagnostic tool for the molecular detection of pathogenic mycobacteria.

Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes.

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.

State-of-the-art detection of Mycobacterium tuberculosis in blood during tuberculosis infection using phage technology.

Tuberculosis (TB), an aerosol-transmitted infection caused by Mycobacterium tuberculosis (Mtb), remains the commonest cause of death globally, from an infectious bacterial disease. Nine years on from the launch of the World Health Organization (WHO)'s END-TB strategy, disease incidence rates are stubbornly unchanged [1]. While this represents, in part, a reversal of improving trends caused by the COVID-19 pandemic, it also reflects the fragility and inadequacy of healthcare systems to sustain TB control [2]. Although multifactorial, a key reason for this is the ineffectiveness of existing clinical tools to meet the two key objectives of the END-TB strategy-(i) early diagnosis and treatment of TB disease (to limit onward transmission); and (ii) disease prevention through screening for asymptomatic TB infection (TBI). Meeting both objectives will rely on the development of new biomarkers with high accuracy, but the global nature of the TB problem also requires that new tests are rapid, low cost and can be measured in patients by sampling from universally accessible sites. In this review, we will present the accumulating evidence for circulating Mtb in both TB disease and asymptomatic TBI and discuss the potential utility of novel bacteriophage-based technology for blood-based detection of Mtb.

α-Methylacyl-CoA Racemase from Mycobacterium tuberculosis-Detailed Kinetic and Structural Characterization of the Active Site.

α-Methylacyl-CoA racemase in M. tuberculosis (MCR) has an essential role in fatty acid metabolism and cholesterol utilization, contributing to the bacterium's survival and persistence. Understanding the enzymatic activity and structural features of MCR provides insights into its physiological and pathological significance and potential as a therapeutic target. Here, we report high-resolution crystal structures for wild-type MCR in a new crystal form (at 1.65 Å resolution) and for three active-site mutants, H126A, D156A and E241A, at 2.45, 1.64 and 1.85 Å resolutions, respectively. Our analysis of the new wild-type structure revealed a similar dimeric arrangement of MCR molecules to that previously reported and details of the catalytic site. The determination of the structures of these H126A, D156A and E241A mutants, along with their detailed kinetic analysis, has now allowed for a rigorous assessment of their catalytic properties. No significant change outside the enzymatic active site was observed in the three mutants, establishing that the diminution of catalytic activity is mainly attributable to disruption of the catalytic apparatus involving key hydrogen bonding and water-mediated interactions. The wild-type structure, together with detailed mutational and biochemical data, provide a basis for understanding the catalytic properties of this enzyme, which is important for the design of future anti-tuberculosis drug molecules.

Prevalence of Smear-Positive, Rifampicin-Resistant Mycobacterium tuberculosis and Related Factors Among Residents with Cough in Northern Ethiopian Refugee Health Facilities.

Purpose: To ascertain the prevalence of Mycobacterium tuberculosis (M.tb) among refugees suspected of tuberculosis (TB) and related risk factors, including smear-positive and Rifampicin-resistant M.tb. Methods: A cross-sectional study was conducted between January 2020 and May 2020 among 384 refugees in four refugee camps in Northwest Tigray, Ethiopia. Socio-demographic and clinical data were collected from refugees with a history of cough for more than two weeks prospectively. Spot-spot sputum samples were collected and transported in an ice box to the Shire Suhul Hospital Microbiology laboratory; and then examined using a Fluorescent Microscope. All smear-positive samples were further processed by GeneXpert to detect Rifampicin-resistant MTB. Data were analyzed using SPSS version 21 and a p-value <0.05 was considered statistically significant. Results: The overall prevalence of smear-positive PTB infection was 5.5% (21/384), but No TB case was resistant to Rifampicin detected by GeneXpert MTB/RIF assay. About 70% of the smear-positive pulmonary TB identified were females. Five (23.8%) of the smear-positive pulmonary tuberculosis cases were co-infected by HIV. Sharing of drink and food materials (AOR = 4.36, 95% CI = 1.19-15.89), active TB contact (AOR 7.24, 95% CI = 1.62-32.125), BMI (AOR = 5.23, 95% CI = 1.28-21.29), opening window practice (AOR = 4.32, 95% CI = 1.02-18.30) and HIV status (AOR = 9.36, 95% CI = 1.64-53.35) were statistically significant predisposing factors. Conclusion: The prevalence of smear-positive pulmonary TB among northwest Tigray refugee camps was still high. The prevalence of TB/HIV co-infection was also high. Minimizing close contact with active TB cases, reducing malnutrition, rapid TB/HIV screening, and establishing a ventilation system can reduce the transmission of TB among refugees.

The first insight into Mycobacterium tuberculosis complex isolates in the lower northern region in Thailand.

BACKGROUND: Tuberculosis (TB) remains an important infectious disease and different genotypes have been reported. This study aimed to investigate the genetic diversity and molecular epidemiology of TB in the lower northern region of Thailand, where genotyping data are limited. METHODS: A total of 159 Mycobacterium tuberculosis complex (MTBC) isolates from this region were genotyped by spoligotyping and the major spoligotypes were further subdivided by the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method. RESULTS: Spoligotyping identified 34 types and classified them into 14 clusters. East African-Indian (EAI) groups were the most frequent (44.7%), followed by Beijing (36.5%), with a higher prevalence of drug resistance. By 15-loci MIRU-VNTR typing, the major groups of the Beijing and EAI2_NTB were further differentiated into 44 and 21 subtypes forming 9 and 5 subclusters with cluster rates of 0.26 and 0.44, respectively. The Hunter-Gaston Discriminatory Index among the Beijing and EAI2_NTB groups were 0.987 and 0.931, respectively, indicating high diversity. CONCLUSIONS: This is the first look at the MTBC genotypes in the lower northern region of Thailand, which could aid in understanding the distribution and potential spread of MTBC and Mycobacterium bovis in the target region to support TB control in Thailand.

Negative Regulation of Autophagy during Macrophage Infection by Mycobacterium bovis BCG via Protein Kinase C Activation.

Mycobacterium tuberculosis (Mtb) employs various strategies to manipulate the host's cellular machinery, overriding critical molecular mechanisms such as phagosome-lysosome fusion, which are crucial for its destruction. The Protein Kinase C (PKC) signaling pathways play a key role in regulating phagocytosis. Recent research in Interferon-activated macrophages has unveiled that PKC phosphorylates Coronin-1, leading to a shift from phagocytosis to micropinocytosis, ultimately resulting in Mtb destruction. Therefore, this study aims to identify additional PKC targets that may facilitate Mycobacterium bovis (M. bovis) infection in macrophages. Protein extracts were obtained from THP-1 cells, both unstimulated and mycobacterial-stimulated, in the presence or absence of a general PKC inhibitor. We conducted an enrichment of phosphorylated peptides, followed by their identification through mass spectrometry (LC-MS/MS). Our analysis revealed 736 phosphorylated proteins, among which 153 exhibited alterations in their phosphorylation profiles in response to infection in a PKC-dependent manner. Among these 153 proteins, 55 are involved in various cellular processes, including endocytosis, vesicular traffic, autophagy, and programmed cell death. Importantly, our findings suggest that PKC may negatively regulate autophagy by phosphorylating proteins within the mTORC1 pathway (mTOR2/PKC/Raf-1/Tsc2/Raptor/Sequestosome-1) in response to M. bovis BCG infection, thereby promoting macrophage infection.

Identification of genetic determinants of bedaquiline resistance in Mycobacterium tuberculosis in Ural region, Russia.

Collecting data on rare Mycobacterium tuberculosis (Mtb) clinical isolates with resistance to the new anti-tuberculosis drug bedaquiline is an important task for improving antimicrobial susceptibility testing methods. Nanopore whole genome sequencing, the proportion method on Middlebrook 7H11 medium, and BACTEC MGIT 960 assays were used to analyze genotypic and phenotypic resistance to bedaquiline. We found four mutations: atpE I66M, atpE А63Р, Rv0678 А36Т, and Rv0678 S53P in five isolates with different levels of phenotypic bedaquiline resistance. IMPORTANCE: Bedaquiline (BDQ) is a new anti-tuberculosis drug. The phenotypic and genotypic data describing the mechanism of drug resistance are critical for the design of rapid and accurate diagnostic tests. We consider that our work, which describes genotypic and phenotypic resistance to BDQ, can contribute to the standardization of drug susceptibility testing.

Molecular detection and characterization of the Mycobacterium tuberculosis complex subspecies responsible for bovine tuberculosis in Punjab, Pakistan.

Bovine tuberculosis (bTB), traditionally associated with Mycobacterium bovis, presents significant public health and economic challenges worldwide. This study investigated the causative agents of bTB in slaughtered cattle and buffalo in Lahore, Pakistan. Of the 3,581 animals screened, 34 were identified with gross TB-like lesions. The lesions were processed for culture, PCR, and Sanger sequencing to identify the causative agents of the disease. The results identified 10 Mycobacterium orygis and 8 Mycobacterium tuberculosis sensu stricto isolates. Whole-genome sequencing was performed on two M. orygis isolates, and the sequences were phylogenetically compared to 93 publicly available M. orygis sequences. The results also demonstrated that the JB21 and JB22 primers, which have been previously commonly applied to detect M. bovis in Pakistan, are unable to distinguish between M. tuberculosis complex subspecies. The identification of M. orygis and M. tuberculosis as causative agents of bTB in this slaughterhouse in Punjab may have important implications in identifying cases of zoonotic TB in humans and applying appropriate molecular tools to identify the prevalence of the disease. The data from this study align with recent findings suggesting M. orygis is the predominant cause of bTB in South Asia.IMPORTANCEThe study findings hold significant relevance to the Journal of Clinical Microbiology, as they directly impact the field. The first-time identification of Mycobacterium orygis and Mycobacterium tuberculosis as the predominant causative agents of bovine tuberculosis in Lahore, Pakistan underscores the urgent need for enhanced diagnostic methods. The study emphasizes the importance of improved assays for the accurate detection and differentiation of Mycobacterium subspecies. Additionally, the research addresses zoonotic risk assessment and public health implications, advocating for a multidisciplinary approach that integrates clinical microbiology with veterinary and human health sectors. These insights contribute to clinical microbiology knowledge, shaping effective strategies for disease prevention, surveillance, and control. The study's potential to advance the field makes it well suited for publication in the Microbiology Spectrum journal.

Bacterial pathogens in Xpert MTB/RIF Ultra-negative sputum samples of patients with presumptive tuberculosis in a high TB burden setting: a 16S rRNA analysis.

In patients with presumptive tuberculosis (TB) in whom the diagnosis of TB was excluded, understanding the bacterial etiology of lower respiratory tract infections (LRTIs) is important for optimal patient management. A secondary analysis was performed on a cohort of 250 hospitalized patients with symptoms of TB. Bacterial DNA was extracted from sputum samples for Illumina 16S rRNA sequencing to identify bacterial species based on amplicon sequence variant level. The bacterial pathogen most likely to be responsible for the patients' LRTI could only be identified in a minority (6.0%, 13/215) of cases based on 16S rRNA amplicon sequencing: Mycoplasma pneumoniae (n = 7), Bordetella pertussis (n = 2), Acinetobacter baumanii (n = 2), and Pseudomonas aeruginosa (n = 2). Other putative pathogens were present in similar proportions of Xpert Ultra-positive and Xpert Ultra-negative sputum samples. The presence of Streptococcus (pseudo)pneumoniae appeared to increase the odds of radiological abnormalities (aOR 2.5, 95% CI 1.12-6.16) and the presence of S. (pseudo)pneumoniae (aOR 5.31, 95% CI 1.29-26.6) and Moraxella catarrhalis/nonliquefaciens (aOR 12.1, 95% CI 2.67-72.8) increased the odds of 6-month mortality, suggesting that these pathogens might have clinical relevance. M. pneumoniae, B. pertussis, and A. baumanii appeared to be the possible causes of TB-like symptoms. S. (pseudo)pneumoniae and M. catarrhalis/nonliquefaciens also appeared of clinical relevance based on 16S rRNA amplicon sequencing. Further research using tools with higher discriminatory power than 16S rRNA sequencing is required to develop optimal diagnostic and treatment strategies for this population.IMPORTANCEThe objective of this study was to identify possible bacterial lower respiratory tract infection (LRTI) pathogens in hospitalized patients who were initially suspected to have TB but later tested negative using the Xpert Ultra test. Although 16S rRNA was able to identify some less common or difficult-to-culture pathogens such as Mycoplasma pneumoniae and Bordetella pertussis, one of the main findings of the study is that, in contrast to what we had hypothesized, 16S rRNA is not a method that can be used to assist in the management of patients with presumptive TB having a negative Xpert Ultra test. Even though this could be considered a negative finding, we believe it is an important finding to report as it highlights the need for further research using different approaches.

Mycobacterium tuberculosis Antigen 85B modifies BCG-induced anti-tuberculosis immunity and favors pathogen survival.

Tuberculosis is one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis (Mtb) has developed strategies not only to evade from host immunity but also to manipulate it for its survival. We investigated whether Mtb exploited the immunogenicity of Ag85B, one of its major secretory proteins, to redirect host anti-TB immunity to its advantage. We found that administration of Ag85B protein to mice vaccinated with Bacillus Calmette-Guérin (BCG) impaired the protection elicited by vaccination causing a more severe infection when mice were challenged with Mtb. Ag85B administration reduced BCG-induced CD4 T cell activation and IFN-γ, CCL-4 and IL-22 production in response to Mtb-infected cells. On the other hand, it promoted robust Ag85B-responsive IFN-γ-producing CD4 T cells, expansion of a subset of IFN-γ/IL-10-producing CD4+FOXP3+Treg cells, differential activation of IL-17/IL-22 responses and activation of regulatory and exhaustion pathways, including programmed death-ligand 1 expression on macrophages. All this resulted in impaired intracellular Mtb growth control by systemic immunity, both at pre- and post-Mtb challenge. Interestingly, Mtb infection itself generated Ag85B-reactive inflammatory immune cells incapable of clearing Mtb in both unvaccinated and BCG-vaccinated mice. Our data suggest that Mtb can exploit the strong immunogenicity of Ag85B to promote its own survival and spread. Since Ag85B is normally secreted by replicating bacteria and it is commonly found in the lungs of the Mtb-infected host, our findings may advance the understanding on the mechanisms of Mtb pathogenesis and immune evasion.

Antimycobacterial potential of Trachyspermum ammi seed essential oil via fume contact and determination of major compounds.

Trachyspermum ammi (L.), commonly known as carrom seeds or Ajwain, has been extensively studied for its medicinal properties. In this study, anti-mycobacterial effect of AEO in liquid and fume form was investigated against Mycobacterium smegmatis and Mycobacterium tuberculosis (M. tb). Results showed that AEO inhibits the growth of M. smegmatis at 0.03 mg/mL and becomes bactericidal at 0.125 mg/mL. MICs were observed at 0.03, 0.125 and 0.06 mg/mL against M. tb (H37Rv), isoniazid- and rifampicin-resistant (RIF-R) strains. Inverted disc-fume assay revealed AEO and Thymol efficiently inhibit the growth of M. smegmatis and M. tb. Similarly, in fume contact AEO and Thymol demonstrated antibiofilm activity at a dose of 1.25 mg/mL air and 40 mg/mL air against M.smegmatis effectively. GC-MS analysis showed that Thymol was the dominant compound. These findings suggest that the use of AEO in fume form may serve as a promising strategy as an anti-mycobacterial activity against M. tb.

Bioisosteric modification of Linezolid identified the potential M. tuberculosis protein synthesis inhibitors to overcome the myelosuppression and serotonergic toxicity associated with Linezolid in the treatment of the multi-drug resistance tuberculosis (MDR-TB).

Linezolid is the first and only oxazolidinone antibacterial drug was approved in the last 35 years. It exhibits bacteriostatic efficacy against M. tuberculosis and is a crucial constituent of the BPaL regimen (Bedaquiline, Pretomanid, and Linezolid), which was authorized by the FDA in 2019 for the treatment of XDR-TB or MDR-TB. Despite its unique mechanism of action, Linezolid carries a considerable risk of toxicity, including myelosuppression and serotonin syndrome (SS), which is caused by inhibition of mitochondrial protein synthesis (MPS) and monoamine oxidase (MAO), respectively. Based on the structure toxicity relationship (STR) of Linezolid, in this work, we used a bioisosteric replacement approach to optimize the structure of Linezolid at the C-ring and/or C-5 position for myelosuppression and serotogenic toxicity. Extensive hierarchical multistep docking, drug likeness prediction, molecular binding interactions analyses, and toxicity assessment identified three promising compounds (3071, 7549 and 9660) as less toxic potential modulators of Mtb EthR protein. Compounds 3071, 7549 and 9660 were having the significant docking score of -12.696 Kcal/mol, -12.681 Kcal/mol and -15.293 Kcal/mol towards the Mtb EthR protein with less MAO-A and B affinity [compound 3071: MAO A (-4.799 Kcal/mol) and MAO B (-6.552 Kcal/mol); compound 7549: MAO A (> -2.00 Kcal/mol) and MAO B (> -2.00 Kcal/mol) and compound 9660: MAO A (> -5.678 Kcal/mol) and MAO B (> -6.537Kcal/mol) and none of them shown the Leukopenia as a side effect due to the Myelosuppression. The MD simulation results and binding free energy estimations correspond well with docking analyses, indicating that the proposed compounds bind and inhibit the EthR protein more effectively than Linezolid. The quantum mechanical and electrical characteristics were evaluated using density functional theory (DFT), which also demonstrated that the proposed compounds are more reactive than Linezolid.Communicated by Ramaswamy H. Sarma.

Phenotype versus genotype discordant rifampicin susceptibility testing in tuberculosis: implications for a diagnostic accuracy.

IMPORTANCE: An accurate diagnosis of drug resistance in clinical isolates is an important step for better treatment outcomes. The current study observed a higher discordance rate of rifampicin resistance on Mycobacteria Growth Indicator Tube (MGIT) drug susceptibility testing (DST) than Lowenstein-Jenson (LJ) DST when compared with the rpoB sequencing. We detected a few novel mutations and their combination in rifampicin resistance isolates that were missed by MGIT DST and may be useful for the better management of tuberculosis (TB) treatment outcomes. Few novel deletions in clinical isolates necessitate the importance of rpoB sequencing in large data sets in geographic-specific locations, especially high-burden countries. We explored the discordance rate on MGIT and LJ, which is important for the clinical management of rifampicin resistance to avoid the mistreatment of drug-resistant TB. Furthermore, MGIT-sensitive isolates may be subjected to molecular methods of diagnosis for further confirmation and treatment options.

Elucidating specific interactions for designing novel pyrrolamide derivatives as potential GyrB inhibitors based on ab initio fragment molecular orbital calculations.

Tuberculosis (TB), the second leading infectious killer, causes serious public health problems worldwide. To develop novel anti-TB agents, many biochemical studies have targeted the subunit B of DNA gyrase (GyrB), which captures a second DNA segment and responses for ATP hydrolysis. Here, we investigated specific interactions between GyrB residues and existing pyrrolamide derivatives at an electronic level using ab initio fragment molecular orbital (FMO) calculations and designed potent inhibitors against GyrB. The evaluated binding affinities between GyrB and pyrrolamides were confirmed to be consistent with the IC50 values obtained from previous experiments. Thus, we employed the most potent pyrrolamide (compound 1) as a lead compound and proposed novel pyrrolamide derivatives. The specific interactions between GyrB and these derivatives were investigated using molecular mechanic optimizations and FMO calculations. The results revealed that our proposed derivatives had strong hydrogen bonds with Asp79 and Arg141 and exhibited electrostatic interactions with Glu56 and Ile84 of GyrB. In addition, the binding affinity between GyrB and compound 1 was enhanced significantly by the replacement at the R3 site of compound 1. The present results may provide structural concepts for the rational design of potent GyrB inhibitors as anti-TB agents.Communicated by Ramaswamy H. Sarma.

Whole genome sequencing reveals candidate genes involving in PAS resistance in M. Tuberculosis isolated from patients in Thailand.

Para-amino salicylic acid (PAS) was first reported by Lehmann in 1946 and used for tuberculosis treatment. However, due to its adverse effects, it is now used only as a second line anti-tuberculosis drug for treatment of multidrug resistant or extensively drug resistant M. tuberculosis. The structure of PAS is similar to para-amino benzoic acid (pABA), an intermediate metabolite in the folate synthesis pathway. The study has identified mutations in genes in folate pathway and their intergenic regions for their possibilities in responsible for PAS resistance. Genomic DNA from 120 PAS-resistant and 49 PAS-sensitive M. tuberculosis isolated from tuberculosis patients in Thailand were studied by whole genome sequencing. Twelve genes in the folate synthesis pathway were investigated for variants associated with PAS resistance. Fifty-one SNVs were found in nine genes and their intergenic regions (pabC, pabB, folC, ribD, thyX, dfrA, thyA, folK, folP). Functional correlation test confirmed mutations in RibD, ThyX, and ThyA are responsible for PAS resistance. Detection of mutation in thyA, folC, intergenic regions of thyX, ribD, and double deletion of thyA dfrA are proposed for determination of PAS resistant M. tuberculosis.

A secreted form of chorismate mutase (Rv1885c) in Mycobacterium bovis BCG contributes to pathogenesis by inhibiting mitochondria-mediated apoptotic cell death of macrophages.

BACKGROUND: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown. METHODS: A tbcm deletion mutant (B∆tbcm) was generated by targeted gene knockout in BCG to investigate the pathogenic role of TBCM in mice or macrophages. We compared the pathogenesis of B∆tbcm and wild-type BCG in vivo by measuring the bacterial clearance rate and the degree of apoptosis. Promotion of the intrinsic apoptotic pathway was evaluated in infected bone marrow-derived macrophages (BMDMs) by measuring apoptotic cell death, loss of mitochondrial membrane potential and translocation of pore-forming proteins. Immunocytochemistry, western blotting and real-time PCR were also performed to assess the related protein expression levels after infection. Furthermore, these findings were validated by complementation of tbcm in BCG. RESULTS: Deletion of the tbcm gene in BCG leads to reduced pathogenesis in a mouse model, compared to wild type BCG, by promoting apoptotic cell death and bacterial clearance. Based on these findings, we found that intrinsic apoptosis and mitochondrial impairment were promoted in B∆tbcm-infected BMDMs. B∆tbcm down-regulates the expression of Bcl-2, which leads to mitochondrial outer membrane permeabilization (MOMP), culminating in cytochrome c release from mitochondria. Consistent with this, transcriptome profiling also indicated that B∆tbcm infection is more closely related to altered mitochondrial-related gene expression than wild-type BCG infection, suggesting an inhibitory role of TBCM in mitochondrial dysfunction. Moreover, genetic complementation of B∆tbcm (C∆tbcm) restored its capacity to inhibit mitochondria-mediated apoptotic cell death. CONCLUSIONS: Our findings demonstrate the contribution of TBCM to bacterial survival, inhibiting intrinsic apoptotic cell death of macrophages as a virulence factor of M. tuberculosis complex (MTBC) strains, which could be a potential target for the development of TB therapy.