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This study reports the synthesis and evaluation of a series of new pyridazin-3-ones with furan moieties 5a–j and 6a–f, to test for their antimycobacterial and antifungal activities. The structures of the target compounds were confirmed by elemental analysis and spectroscopic techniques (IR, mass, 1H-and 13C-NMR). Amongst the compounds tested, 5e, 5g, 5i and 6e exhibited highest activity against Mycobacterium tuberculosis, while 5h, 6d and 6f showed moderate in vitro antifungal activities against Candida albicans and Candida parapsilosis.

The natural bioactive products myxin and iodinin are phenazine 5,10-dioxides possessing potent anti-bacterial and anti-cancer activity in vitro. This work describes the synthesis and derivatization of new myxin and iodinin regioisomers, developed from 1,3-dihydroxyphenazine 5,10-dioxide. Compounds were evaluated for activity towards M. tuberculosis (Mtb) strains, a human AML cell line (MOLM-13), and two non-cancerous mammalian cell lines (NRK and H9c2). Highly potent analogs were developed having IC 50 values against MTB down to 20 nM and 1.4 μM for human AML cells. 1-OH-3-O-alkyl substituted derivatives demonstrated high efficacy against Mtb and low toxicity in normal cells. 2,3-substituted regioisomers of myxin and iodinin were shown to be inactive, highlighting the importance of oxygen substituent in position 1 of the scaffold. A strong positive correlation between anti-MTB and anti-AML activity was revealed, suggesting a common mechanism of action in bacteria and cancer cells. These findings demonstrate the therapeutic potential of 1,3-O-functionalized phenazine 5,10-dioxides in chemotherapy for Mtb and AML and contribute to the structure-activity understanding of phenazine 5,10-dioxides with respect to their biological activity., Competing Interests: Declaration of competing interest PR and EÖV are among inventors of a patent No.: US 11,958,842 B2. Current assigne is Adjutec Pharma AS, Norway. PR is an employe of Adjutec Pharma., (Copyright © 2025 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Introduction: Despite being implicated in a wide spectrum of community- and healthcare-acquired infections, anaerobes have not yet been incorporated into systematic surveillance programs in Europe., Methods: We conducted a multicentre retrospective observational study analysing all anaerobic strains isolated from blood cultures in 44 European Hospital Centres over a 4-year period (2020-2023). Diagnostic approach, epidemiology, and antimicrobial susceptibility according to EUCAST v. 15.0 were investigated., Results: Our study included 14,527 anaerobes, most of which were Gram-positive (45%) or Gram-negative (40%) bacilli. MALDI-TOF coupled to mass spectrometry was the most widely used tool for species identification (98%). Antimicrobial susceptibility testing was performed in the vast majority of centres, using mostly gradient diffusion strip (77%) and disk diffusion (45%) methods according to EUCAST guidelines (v. 15.0). The most prevalent species were Cutibacterium acnes (18.7%), Bacteroides fragilis (16.3%), Clostridium perfringens (5.3%), Bacteroides thetaiotaomicron (4.2%), Fusobacterium nucleatum (3.5%), and Parvimonas micra (3.4%). C. acnes showed high resistance to benzylpenicillin (18%), clindamycin (39%), and imipenem (19% and 13% by MIC methods and disk diffusion, respectively). B. fragilis showed high resistance to amoxicillin/clavulanate (24%), piperacillin/tazobactam (22% and 14% by MIC methods and disk diffusion, respectively), clindamycin (22% by both MIC methods and disk diffusion), meropenem (13%), and metronidazole (10%, only by disk diffusion). A similar resistance pattern was observed in B. thetaiotaomicron, Bacteroides ovatus, and Parabacteroides distasonis. C. perfringens showed high resistance to clindamycin (69% and 45% by MIC methods and disk diffusion, respectively), while benzylpenicillin and metronidazole maintained over 90% activity. F. nucleatum showed high resistance to benzylpenicillin (11%), while Fusobacterium necrophorum showed alarming rates of resistance to clindamycin (12%), meropenem (16%) and metronidazole (11%)., Conclusions: This study presented an up-to-date analysis of the diagnostics and epidemiology of anaerobic bacteria in Europe, providing insights for future comparative analyses and the development of antimicrobial diagnostic and management strategies, as well as the optimization of current antibiotic treatments., Competing Interests: Competing Interests None., (Copyright © 2025. Published by Elsevier Ltd.)

Background: Human macrophages generate antimicrobial reactive nitrogen species in response to infection by Mycobacterium tuberculosis (Mtb). Exposure to these redox-reactive compounds induces stress response in Mtb, which can affect posttranslational modifications (PTM)., Methods: Here, we present the global analysis of the PTM acylation of Mtb proteins in response to a sublethal dose of nitrosative stress in the form of nitric oxide (NO) using label free quantification., Results: A total of 6437 acylation events were identified on 1496 Mtb proteins, and O-acylation accounted for 92.2% of the events identified, while 7.8% were N-acylation events. About 22% of the sites identified were found to be acylated by more than one acyl-group. Furthermore, the abundance of each acyl-group decreased as their molecular weight increased. Quantitative PTM analysis revealed differential abundance of acylation in proteins involved in stress response, iron ion homeostasis, growth, energy metabolism, and antimicrobial resistance (AMR) induced by nitrosative stress over time., Conclusions: The results reveal a potential role of Mtb protein acylation in the bacterial stress responses and AMR. To our knowledge, this is the first report on global O-acylation profile of Mtb in response to NO. This will significantly improve our understanding of the changes in Mtb acylation under nitrosative stress, highly relevant for global health., (© 2024 The Author(s). PROTEOMICS ‐ Clinical Applications published by Wiley‐VCH GmbH.)

Mycobacterium abscessus ( M. abscessus ) is a multidrug-resistant nontuberculous mycobacterium (NTM) that is responsible for a wide spectrum of infections in humans. The lack of effective bactericidal drugs and the formation of biofilm make its clinical treatment very difficult. The FDA-approved drug library containing 3048 marketed and pharmacopeial drugs or compounds was screened at 20 μM against M. abscessus type strain 19977 in 7H9 medium, and 62 hits with potential antimicrobial activity against M. abscessus were identified. Among them, bithionol, a clinically approved antiparasitic agent, showed excellent antibacterial activity and inhibited the growth of three different subtypes of M. abscessus from 0.625 μM to 2.5 μM. We confirmed the bactericidal activity of bithionol by the MBC/MIC ratio being ≤4 and the time-kill curve study and also electron microscopy study. Interestingly, it was found that at 128 μg/mL, bithionol could completely eliminate biofilms after 48h, demonstrating an outstanding antibiofilm capability compared to commonly used antibiotics. Additionally, bithionol could eliminate 99.9% of biofilm bacteria at 64 μg/mL, 99% at 32 μg/mL, and 90% at 16 μg/mL. Therefore, bithionol may be a potential candidate for the treatment of M. abscessus infections due to its significant antimicrobial and antibiofilm activities.

Serotonergic toxicity due to MAO enzyme inhibition is a significant concern when using linezolid to treat MDR-TB. To address this issue, we designed linezolid bioisosteres with a modified acetamidomethyl side chain at the C-5 position of the oxazolidine ring to balance activity and reduce toxicity. Among these bioisosteres, R7 emerged as a promising candidate, demonstrating greater effectiveness against M. tuberculosis ( Mtb ) H 37 Rv cells with an MIC of 2.01 μM compared to linezolid (MIC = 2.31 μM). Bioisostere R7 also exhibited remarkable activity (MIC 50 ) against drug-resistant Mtb clinical isolates, with values of 0.14 μM (INH R , inhA+ ), 0.53 μM (INH R , katG+ ), 0.24 μM (RIF R , rpoB+ ), and 0.92 μM (INH R INH R , MDR). Importantly, it was >6.52 times less toxic as compared to the linezolid toward the MAO-A and >64 times toward the MAO-B enzyme, signifying a substantial improvement in its drug safety profile., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

A new class of structurally intriguing heterocycles embedded with spiropyrrolidine, oxindole and chromanones was prepared by regio- and stereoselectively in quantitative yields using an intermolecular tandem cycloaddition protocol. The compounds synthesized were assayed for their anti-mycobacterial activity against Mycobacterium tuberculosis ( Mtb ) H37Rv and isoniazid-resistant ( katG and inhA promoter mutations) clinical Mtb isolates. Four compounds exhibited significant antimycobacterial activity against Mtb strains tested. In particular, a compound possessing a fluorine substituted derivative displayed potent activity at 0.39 μg mL -1 against H37Rv, while it showed 0.09 μg mL -1 and 0.19 μg mL -1 activity against inhA promoter and katG mutation isolates, respectively. A molecular docking study was conducted with the potent compound, which showed results that were consistent with the in vitro experiments., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Purpose: To identify essential oils (EOs) active against non-growing stationary phase Mycobacterium abscessus and multidrug-resistant M. abscessus strains., Methods: The activity of EOs against both stationary and log phase M. abscessus was evaluated by colony forming unit (CFU) assay and minimum inhibitory concentration (MIC) testing., Results: We assessed the activity of 80 EOs against stationary phase M. abscessus and found 12 EOs (Cinnamon, Satureja montana, Palmarosa, Lemon eucalyptus, Honey myrtle, Combava, Health shield, Mandarin, Thyme, Rosewood, Valerian Root and Basil) at 0.5% concentration to be active against both growing and non-growing stationary phase M. abscessus. Among them, Satureja montana essential oil and Palmarosa essential oil could eliminate all stationary phase M. abscessus at 0.125% and Cinnamon essential oil could eliminate stationary phase bacteria at 0.063% after 1-day treatment. Interestingly, these EOs also exhibited promising activity against multidrug-resistant M. abscessus clinical strains., Conclusions: Our study indicates that some EOs display outstanding effectiveness against both drug susceptible M. abscessus and multidrug-resistant M. abscessus isolates. These findings may be significant for the treatment of persistent M. abscessus infections., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Serotogenic toxicity is a major hurdle associated with Linezolid in the treatment of drug-resistant tuberculosis (TB) due to the inhibition of monoamine oxidase (MAO) enzymes. Azole compounds demonstrate structural similarities to the recognized anti-TB drug Linezolid, making them intriguing candidates for repurposing. Therefore, we have repurposed azoles (Posaconazole, Itraconazole, Miconazole, and Clotrimazole) for the treatment of drug-resistant TB with the anticipation of their selectivity in sparing the MAO enzyme. The results of repurposing revealed that Clotrimazole showed equipotent activity against the Mycobacterium tuberculosis ( Mtb ) H 37 Rv strain compared to Linezolid, with a minimal inhibitory concentration (MIC) of 2.26 μM. Additionally, Clotrimazole exhibited reasonable MIC 50 values of 0.17 μM, 1.72 μM, 1.53 μM, and 5.07 μM against the inhA promoter+, katG+ , rpoB+ , and MDR clinical Mtb isolates, respectively, compared to Linezolid. Clotrimazole also exhibited 3.90-fold less inhibition of MAO-A and 50.35-fold less inhibition of MAO-B compared to Linezolid, suggesting a reduced serotonergic toxicity burden., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

The discovery of new antimicrobial agents as a means of treating drug-resistant microbial pathogens is of utmost significance to overcome their immense risk to human well-being. The current investigation involves the development, synthesis, and assessment of the antimicrobial efficacy of novel quinoline derivatives incorporating a thiosemicarbazide functionality. To design the target compounds ( QST1 - QST14 ), we applied the molecular hybridization approach to link various thiosemicarbazides to the quinoline core with a sulfonyl group. Upon the synthesis and completion of structural characterization via spectroscopic techniques ( 1 H NMR, 13 C NMR, 15 N NMR, IR, and HRMS), the title molecules were extensively evaluated for their potential antitubercular, antibacterial, and antifungal activities. N- (3-Chlorophenyl)-2-(quinolin-8-ylsulfonyl)hydrazine-1-carbothioamide ( QST4 ), the most effective compound against Mycobacterium tuberculosis H37Rv, was also tested on isoniazid-resistant clinical isolates with katG and inhA promoter mutations. Based on molecular docking studies, QST4 was also likely to demonstrate its antimycobacterial activity through inhibition of the InhA enzyme. Furthermore, three derivatives ( QST3 , QST4 , and QST10) with preferable antimicrobial and drug-like profiles were also shown to be nontoxic against human embryonic kidney (HEK) cells. All compounds were optimized by the density functional theory method using B3LYP with the 6-31+G(d,p) basis set. Structural analysis, natural bond orbital calculations of donor-acceptor interactions, molecular electrostatic potential analysis, and frontier molecular orbital analysis were carried out. Quantum chemical descriptors and charges on the atoms were determined to compare the strengths of the intramolecular hydrogen bonds formed and their stabilities. We determined that the sulfur atom forms a stronger intramolecular hydrogen bond than the nitrogen, oxygen, and fluorine atoms in these sulfonyl thiosemicarbazide derivatives., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Mycobacterium tuberculosis ( Mtb ) drug resistance poses an alarming threat to global tuberculosis control. We previously reported that C10 , a ring-fused thiazolo-2-pyridone, inhibits Mtb respiration, blocks biofilm formation, and restores the activity of the antibiotic isoniazid (INH) in INH-resistant Mtb isolates. This discovery revealed a new strategy to address INH resistance. Expanding upon this strategy, we identified C10 analogues with improved potency and drug-like properties. By exploring three heterocycle spacers (oxadiazole, 1,2,3-triazole, and isoxazole) on the ring-fused thiazolo-2-pyridone scaffold, we identified two novel isoxazoles, 17h and 17j . 17h and 17j inhibited Mtb respiration and biofilm formation more potently with a broader therapeutic window, were better potentiators of INH-mediated inhibition of an INH-resistant Mtb mutant, and more effectively inhibited intracellular Mtb replication than C10 . The (-)17j enantiomer showed further enhanced activity compared to its enantiomer and the 17j racemic mixture. Our potent second-generation C10 analogues offer promise for therapeutic development against drug-resistant Mtb .

Isoniazid (INH) is one of the key molecules employed in the treatment of tuberculosis (TB), the most deadly infectious disease worldwide. However, the efficacy of this cornerstone drug has seriously decreased due to emerging INH-resistant strains of Mycobacterium tuberculosis ( Mtb ). In the present study, we aimed to chemically tailor INH to overcome this resistance. We obtained thirteen novel compounds by linking INH to in-house synthesized sulfonate esters via a hydrazone bridge ( SIH1-SIH13 ). Following structural characterization by FTIR, 1 H NMR, 13 C NMR, and HRMS, all compounds were screened for their antitubercular activity against Mtb H37Rv strain and INH-resistant clinical isolates carrying katG and inhA mutations. Additionally, the cytotoxic effects of SIH1-SIH13 were assessed on three different healthy host cell lines; HEK293, IMR-90, and BEAS-2B. Based on the obtained data, the synthesized compounds appeared as attractive antimycobacterial drug candidates with low cytotoxicity. Moreover, the stability of the hydrazone moiety in the chemical structure of the final compounds was confirmed by using UV/Vis spectroscopy in both aqueous medium and DMSO. Subsequently, the compounds were tested for their inhibitory activities against enoyl acyl carrier protein reductase (InhA), the primary target enzyme of INH. Although most of the synthesized compounds are hosted by the InhA binding pocket, SIH1-SIH13 do not primarily show their antitubercular activities by direct InhA inhibition. Finally, in silico determination of important physicochemical parameters of the molecules showed that SIH1-SIH13 adhered to Lipinski's rule of five. Overall, our study revealed a new strategy for modifying INH to cope with the emerging drug-resistant strains of Mtb .

The recent emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) has complicated and significantly slowed efforts to eradicate and/or reduce the worldwide incidence of life-threatening acute and chronic cases of tuberculosis. To overcome this setback, researchers have increased the intensity of their work to identify new small-molecule compounds that are expected to remain efficacious antimicrobials against Mtb. Here, we describe our effort to apply the principles of molecular hybridization to synthesize 16 compounds carrying thiophene and thiazole rings beside the core urea functionality (TTU1-TTU16). Following extensive structural characterization, the obtained compounds were initially evaluated for their antimycobacterial activity against Mtb H37Rv. Subsequently, three derivatives standing out with their anti-Mtb activity profiles and low cytotoxicity (TTU5, TTU6, and TTU12) were tested on isoniazid-resistant clinical isolates carrying katG and inhA mutations. Additionally, due to their pharmacophore similarities to the well-known InhA inhibitors, the molecules were screened for their enoyl acyl carrier protein reductase (InhA) inhibitory potentials. Molecular docking studies were performed to support the experimental enzyme inhibition data. Finally, drug-likeness of the selected compounds was established by theoretical calculations of physicochemical descriptors., (© 2022 Wiley Periodicals LLC.)

Reactive nitrogen species (RNS) are secreted by human cells in response to infection by Mycobacterium tuberculosis (Mtb). Although RNS can kill Mtb under some circumstances, Mtb can adapt and survive in the presence of RNS by a process that involves modulation of gene expression. Previous studies focused primarily on stress-related changes in the Mtb transcriptome. This study unveils changes in the Mtb proteome in response to a sub-lethal dose of nitric oxide (NO) over several hours of exposure. Proteins were identified using liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS/MS). A total of 2911 Mtb proteins were identified, of which 581 were differentially abundant (DA) after exposure to NO in at least one of the four time points (30 min, 2 h, 6 h, and 20 h). The proteomic response to NO was marked by two phases, with few DA proteins in the early phase and a multitude of DA proteins in the later phase. The efflux pump Rv1687 stood out as being the only protein more abundant at all the time points and might play a role in the early protection of Mtb against nitrosative stress. These changes appeared to be compensatory in nature, contributing to iron homeostasis, energy metabolism, and other stress responses. This study thereby provides new insights into the response of Mtb to NO at the level of proteomics., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Background: Tuberculosis, mainly caused by Mycobacterium tuberculosis (Mtb), is an ancient human disease that gravely affects millions of people annually. We wanted to explore the genetic diversity and lineage-specific association of Mtb with drug resistance among pulmonary tuberculosis patients. Methods: Sputum samples were collected from pulmonary tuberculosis patients at six different healthcare institutions in Tigray, Ethiopia, between July 2018 and August 2019. DNA was extracted from 74 Mtb complex isolates for whole-genome sequencing (WGS). All genomes were typed and screened for mutations with known associations with antimicrobial resistance using in silico methods, and results were cross-verified with wet lab methods. Results: Lineage (L) 4 (55.8%) was predominant, followed by L3 (41.2%); L1 (1.5%) and L2 (1.5%) occurred rarely. The most frequently detected sublineage was CAS (38.2%), followed by Ural (29.4%), and Haarlem (11.8%). The recent transmission index (RTI) was relatively low. L4 and Ural strains were more resistant than the other strains to any anti-TB drug ( P < 0.05). The most frequent mutations to RIF, INH, EMB, SM, PZA, ETH, FLQs, and 2nd-line injectable drugs occurred at rpoB S450L, katG S315T, embB M306I/V, rps L K43R, pncA V139A, ethA M1R, gyr A D94G, and rrs A1401G, respectively. Disputed rpoB mutations were also shown in four (16%) of RIF-resistant isolates. Conclusion: Our WGS analysis revealed the presence of diverse Mtb genotypes. The presence of a significant proportion of disputed rpoB mutations highlighted the need to establish a WGS facility at the regional level to monitor drug-resistant mutations. This will help control the transmission of DR-TB and ultimately contribute to the attainment of 100% DST coverage for TB patients as per the End TB strategy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Welekidan, Yimer, Skjerve, Dejene, Homberset, Tønjum and Brynildsrud.)

Biofilm formation is a general strategy for bacterial pathogens to withstand host defense mechanisms. In this study, we found that serum proteases inhibit biofilm formation by Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae , and Bordetella pertussis . Confocal laser-scanning microscopy analysis revealed that these proteins reduce the biomass and alter the architecture of meningococcal biofilms. To understand the underlying mechanism, the serum was fractionated through size-exclusion chromatography and anion-exchange chromatography, and the composition of the fractions that retained anti-biofilm activity against N. meningitidis was analyzed by intensity-based absolute quantification mass spectrometry. Among the identified serum proteins, plasma kallikrein (PKLK), FXIIa, and plasmin were found to cleave neisserial heparin-binding antigen and the α-peptide of IgA protease on the meningococcal cell surface, resulting in the release of positively charged polypeptides implicated in biofilm formation by binding extracellular DNA. Further experiments also revealed that plasmin and PKLK inhibited biofilm formation of B. pertussis by cleaving filamentous hemagglutinin. We conclude that the proteolytic activity of serum proteases toward bacterial adhesins involved in biofilm formation could constitute a defense mechanism for the clearance of pathogens.

Mycobacterium tuberculosis (Mtb), the main etiology of tuberculosis (TB), is predominantly an intracellular pathogen that has caused infection, disease and death in humans for centuries. Lipid droplets (LDs) are dynamic intracellular organelles that are found across the evolutionary tree of life. This review is an evaluation of the current state of knowledge regarding Mtb-LD formation and associated Mtb transcriptome directly from sputa.Based on the LD content, Mtb in sputum may be classified into three groups: LD positive, LD negative and LD borderline. However, the clinical and evolutionary importance of each state is not well elaborated. Mounting evidence supports the view that the presence of LD positive Mtb bacilli in sputum is a biomarker of slow growth, low energy state, towards lipid degradation, and drug tolerance. In Mtb, LD may serve as a source of chemical energy, scavenger of toxic compounds, prevent destruction of Mtb through autophagy, delay trafficking of lysosomes towards the phagosome, and contribute to Mtb persistence. It is suggest that LD is a key player in the induction of a spectrum of phenotypic and metabolic states of Mtb in the macrophage, granuloma and extracellular sputum microenvironment. Tuberculosis patients with high proportion of LD positive Mtb in pretreatment sputum was associated with higher rate of poor treatment outcome, indicating that LD may have a clinical application in predicting treatment outcome.The propensity for LD formation among Mtb lineages is largely unknown. The role of LD on Mtb transmission and disease phenotype (pulmonary TB vs extra-pulmonary TB) is not well understood. Thus, further studies are needed to understand the relationships between LD positivity and Mtb lineage, Mtb transmission and clinical types., (© 2021. The Author(s).)

Objectives: Tuberculosis (TB) is a preventable and treatable infectious disease, but the continuing emergence and spread of multidrug-resistant TB is threatening global TB control efforts. This study aimed to describe the frequency and patterns of drug resistance-conferring mutations of Mycobacterium tuberculosis (MTB) isolates detected from pulmonary TB patients in Tigray Region, Ethiopia., Methods: A cross-sectional study design was employed to collect sputum samples from pulmonary TB patients between July 2018 to August 2019. Culture and identification tests were done at Tigray Health Research Institute (THRI). Mutations conferring rifampicin (RIF), isoniazid (INH) and fluoroquinolone (FQ) resistance were determined in 227 MTB isolates using GenoType MTBDRplus and GenoType MTBDRsl., Results: Mutations conferring resistance to RIF, INH and FQs were detected in 40/227 (17.6%), 41/227 (18.1%) and 2/38 (5.3%) MTB isolates, respectively. The majority of mutations for RIF, INH and FQs occurred at codons rpoB S531L (70%), katG S315T (78%) and gyrA D94Y/N (100%), respectively. This study revealed a significant number of unknown mutations in the rpoB, katG and inhA genes., Conclusion: High rates of mutations conferring resistance to RIF, INH and FQs were observed in this study. A large number of isolates showed unknown mutations, which require further DNA sequencing analysis. Periodic drug resistance surveillance and scaling-up of drug resistance testing facilities are imperative to prevent the transmission of drug-resistant TB in the community., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Despite the discovery of the tubercle bacillus more than 130 years ago, its physiology and the mechanisms of virulence are still not fully understood. A comprehensive analysis of the proteomes of members of the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages 3, 4, 5, and 7 was conducted to better understand the evolution of virulence and other physiological characteristics. Unique and shared proteomic signatures in these modern, pre-modern and ancient MTBC lineages, as deduced from quantitative bioinformatics analyses of high-resolution mass spectrometry data, were delineated. The main proteomic findings were verified by using immunoblotting. In addition, analysis of multiple genome alignment of members of the same lineages was performed. Label-free peptide quantification of whole cells from MTBC lineages 3, 4, 5, and 7 yielded a total of 38,346 unique peptides derived from 3092 proteins, representing 77% coverage of the predicted proteome. MTBC lineage-specific differential expression was observed for 539 proteins. Lineage 7 exhibited a markedly reduced abundance of proteins involved in DNA repair, type VII ESX-3 and ESX-1 secretion systems, lipid metabolism and inorganic phosphate uptake, and an increased abundance of proteins involved in alternative pathways of the TCA cycle and the CRISPR-Cas system as compared to the other lineages. Lineages 3 and 4 exhibited a higher abundance of proteins involved in virulence, DNA repair, drug resistance and other metabolic pathways. The high throughput analysis of the MTBC proteome by super-resolution mass spectrometry provided an insight into the differential expression of proteins between MTBC lineages 3, 4, 5, and 7 that may explain the slow growth and reduced virulence, metabolic flexibility, and the ability to survive under adverse growth conditions of lineage 7., (Copyright © 2020 Yimer, Kalayou, Homberset, Birhanu, Riaz, Zegeye, Lutter, Abebe, Holm-Hansen, Aseffa and Tønjum.)

Alzheimer disease (AD) is a devastating neurological disease associated with progressive loss of mental skills and cognitive and physical functions whose etiology is not completely understood. Here, our goal was to simultaneously uncover novel and known molecular targets in the structured layers of the hippocampus and olfactory bulbs that may contribute to early hippocampal synaptic deficits and olfactory dysfunction in AD mice. Spatially resolved transcriptomics was used to identify high-confidence genes that were differentially regulated in AD mice relative to controls. A diverse set of genes that modulate stress responses and transcription were predominant in both hippocampi and olfactory bulbs. Notably, we identify Bok, implicated in mitochondrial physiology and cell death, as a spatially downregulated gene in the hippocampus of mouse and human AD brains. In summary, we provide a rich resource of spatially differentially expressed genes, which may contribute to understanding AD pathology., Competing Interests: J.L. and J.F.N are scientific advisors at 10x Genomics Inc, which provides commercial barcoded arrays. All other authors declare no competing interests.

Background: Tuberculosis (TB) is among the top 10 causes of mortality and the first killer among infectious diseases worldwide. One of the factors fuelling the TB epidemic is the global rise of multidrug resistant TB (MDR-TB). The aim of this study was to determine the magnitude and factors associated with MDR-TB in the Tigray Region, Ethiopia., Method: This study employed a facility-based cross-sectional study design, which was conducted between July 2018 and August 2019. The inclusion criteria for the study participants were GeneXpert-positive who were not under treatment for TB, PTB patients' ≥15 years of age and who provided written informed consent. A total of 300 participants were enrolled in the study, with a structured questionnaire used to collect data on clinical, sociodemographic and behavioral factors. Sputum samples were collected and processed for acid-fast bacilli staining, culture and drug susceptibility testing. Drug susceptibility testing was performed using a line probe assay. Logistic regression was used to analyze associations between outcome and predictor variables., Results: The overall proportion of MDR-TB was 16.7% (11.6% and 32.7% for new and previously treated patients, respectively). Of the total MDR-TB isolates, 5.3% were pre-XDR-TB. The proportion of MDR-TB/HIV co-infection was 21.1%. A previous history of TB treatment AOR 3.75; 95% CI (0.7-2.24), cigarette smoking AOR 6.09; CI (1.65-2.50) and patients who had an intermittent fever (AOR = 2.54, 95% CI = 1.21-5.4) were strongly associated with MDR-TB development., Conclusions: The magnitude of MDR-TB observed among new and previously treated patients is very alarming, which calls for an urgent need for intervention. The high proportion of MDR-TB among newly diagnosed cases indicates ongoing transmission, which suggests the need for enhanced TB control program performance to interrupt transmission. The increased proportion of MDR-TB among previously treated cases indicates a need for better patient management to prevent the evolution of drug resistance. Assessing the TB control program performance gaps and an optimal implementation of the WHO recommended priority actions for the management of drug-resistant TB, is imperative to help reduce the current high MDR-TB burden in the study region., Competing Interests: The authors have declared that no competing interests exist

Antibiotic resistance is one of the major challenges facing modern medicine worldwide. The past few decades have witnessed rapid progress in our understanding of the multiple factors that affect the emergence and spread of antibiotic resistance at the population level and the level of the individual patient. However, the process of translating this progress into health policy and clinical practice has been slow. Here, we attempt to consolidate current knowledge about the evolution and ecology of antibiotic resistance into a roadmap for future research as well as clinical and environmental control of antibiotic resistance. At the population level, we examine emergence, transmission and dissemination of antibiotic resistance, and at the patient level, we examine adaptation involving bacterial physiology and host resilience. Finally, we describe new approaches and technologies for improving diagnosis and treatment and minimizing the spread of resistance., (© FEMS 2020.)

The molecular epidemiology of Mycobacterium tuberculosis (M. tuberculosis, Mtb) is poorly documented in Ethiopia. The data that exists has not yet been collected in an overview metadata form. Thus, this review summarizes available literature on the genomic diversity, geospatial distribution and transmission patterns of Mtb lineages (L) and sublineages in Ethiopia. Spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR) based articles were identified from MEDLINE via PubMed and Scopus. The last date of article search was done on 12th February 2019. Articles were selected following the PRISMA flow diagram. The proportion of (sub)lineages was summarized at national level and further disaggregated by region. Clustering and recent transmission index (RTI) were determined using metan command and random effect meta-analysis model. The meta-analysis was computed using Stata 14 (Stata Corp. College Station, TX, USA). Among 4371 clinical isolates, 99.5% were Mtb and 0.5% were M. bovis. Proportionally, L4, L3, L1 and L7 made up 62.3%, 21.7%, 7.9% and 3.4% of the total isolates, respectively. Among sublineages, L4.2. ETH/SIT149, L4.10/SIT53, L3. ETH1/SIT25 and L4.6/SIT37 were the leading clustered isolates accounting for 14.4%, 9.7%, 7.2% and 5.5%, respectively. Based on MIRU-VNTR, the rate of clustering was 41% and the secondary case rate from a single source case was estimated at 29%. Clustering and recent transmission index was higher in eastern and southwestern Ethiopia compared with the northwestern part of the country. High level of genetic diversity with a high rate of clustering was noted which collectively mirrored the phenomena of micro-epidemics and super-spreading. The largest set of clustered strains deserves special attention and further characterization using whole genome sequencing (WGS) to better understand the evolution, genomic diversity and transmission dynamics of Mtb., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pyrazinamide (PZA) is a unique frontline drug for shortening tuberculosis (TB) treatment, but its mechanisms of action are elusive. We previously found one PZA-resistant strain that harbors an alanine deletion at position 438 (Δ438A) in RpsA, a target of PZA associated with PZA resistance, but its role in causing PZA resistance has been inconclusive. Here, we introduced the RpsA Δ438A mutation along with the D123A mutation into the Mycobacterium tuberculosis chromosome and demonstrated that these RspA mutations are indeed responsible for PZA resistance., (Copyright © 2019 American Society for Microbiology.)

Multiple regulatory mechanisms including post-translational modifications (PTMs) confer complexity to the simpler genomes and proteomes of Mycobacterium tuberculosis (Mtb). PTMs such as glycosylation play a significant role in Mtb adaptive processes. The glycoproteomic patterns of clinical isolates of the Mycobacterium tuberculosis complex (MTBC) representing the lineages 3, 4, 5 and 7 were characterized by mass spectrometry. A total of 2944 glycosylation events were discovered in 1325 proteins. This data set represents the highest number of glycosylated proteins identified in Mtb to date. O-glycosylation constituted 83% of the events identified, while 17% of the sites were N-glycosylated. This is the first report on N-linked protein glycosylation in Mtb and in Gram-positive bacteria. Collectively, the bulk of Mtb glycoproteins are involved in cell envelope biosynthesis, fatty acid and lipid metabolism, two-component systems, and pathogen-host interaction that are either surface exposed or located in the cell wall. Quantitative glycoproteomic analysis revealed that 101 sites on 67 proteins involved in Mtb fitness and survival were differentially glycosylated between the four lineages, among which 64% were cell envelope and membrane proteins. The differential glycosylation pattern may contribute to phenotypic variabilities across Mtb lineages. The study identified several clinically important membrane-associated glycolipoproteins that are relevant for diagnostics as well as for drug and vaccine discovery.

The spread of antibiotic resistance within and between different bacterial populations is a major health problem on a global scale. The identification of genetic transformation in genomic data from Neisseria meningitidis, the meningococcus (Mc), and other bacteria is problematic, since similar or even identical alleles may be involved. A particular challenge in naturally transformable bacteria generally is to distinguish between common ancestry and true recombined sites in sampled genome sequences. Furthermore, the identification of recombination following experimental transformation of homologous alleles requires identifiable differences between donor and recipient, which in itself influences the propensity for homologous recombination (HR). This study identifies the distribution of HR events following intraspecies and interspecies Mc transformations of rpoB alleles encoding rifampicin resistance by whole-genome DNA sequencing and single nucleotide variant analysis. The HR events analysed were confined to the genomic region surrounding the single nucleotide genetic marker used for selection. An exponential length distribution of these recombined events was found, ranging from a few nucleotides to about 72 kb stretches. The lengths of imported sequences were on average found to be longer following experimental transformation of the recipient with genomic DNA from an intraspecies versus an interspecies donor (P<0.001). The recombination events were generally observed to be mosaic, with donor sequences interspersed with recipient sequence. Here, we present four models to explain these observations, by fragmentation of the transformed DNA, by interruptions of the recombination mechanism, by secondary recombination of endogenous self-DNA, or by repair/replication mechanisms.

Age-related changes are increased in patients with Alzheimer's disease (AD), including oxidative stress and DNA damage. We propose that genotoxic stress and DNA repair responses influence neurodegeneration in the pathogenesis of AD. Here, we focus on nucleotide excision repair (NER). Real-time qPCR and mass spectrometry were employed to determine the expression levels of selected NER components. The mRNA levels of the genes encoding the NER proteins RAD23B, RPA1, ERCC1, PCNA and LIG3 as well as the NER-interacting base excision repair protein MPG in blood and brain tissue from four brain regions in patients with AD or mild cognitive impairment and healthy controls (HC), were assessed. NER mRNA levels were significantly higher in brain tissue than in blood. Further, LIG3 mRNA levels in the frontal cortex was higher in AD versus HC, while mRNA levels of MPG and LIG3 in entorhinal cortex and RPA1 in the cerebellum were lower in AD versus HC. In blood, RPA1 and ERCC1 mRNA levels were lower in AD patients than in HC. Alterations in gene expression of NER components between brain regions were associated with AD, connecting DNA repair to AD pathogenesis and suggesting a distinct role for NER in the brain., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The current molecular understanding of Alzheimer's disease (AD) has still not resulted in successful interventions. Mitochondrial dysfunction of the AD brain is currently emerging as a hallmark of this disease. One mitochondrial function often affected in AD is oxidative phosphorylation responsible for ATP production, but also for production of reactive oxygen species (ROS) and for the de novo synthesis of pyrimidines. This paper reviews the role of mitochondrial produced ROS and pyrimidines in the aetiology of AD and their proposed role in oxidative degeneration of macromolecules, synthesis of essential phospholipids and maintenance of mitochondrial viability in the AD brain., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Neisseria meningitidis (Nm) is a Gram-negative nasopharyngeal commensal that can cause septicaemia and meningitis. The neisserial DNA damage-inducible protein DinG is a helicase related to the mammalian helicases XPD and FANCJ. These helicases belong to superfamily 2, are ATP dependent and exert 5' → 3' directionality. To better understand the role of DinG in neisserial genome maintenance, the Nm DinG (DinGNm) enzymatic activities were assessed in vitro and phenotypical characterization of a dinG null mutant (NmΔdinG) was performed. Like its homologues, DinGNm possesses 5' → 3' directionality and prefers DNA substrates containing a 5'-overhang. ATPase activity of DinGNm is strictly DNA-dependent and DNA unwinding activity requires nucleoside triphosphate and divalent metal cations. DinGNm directly binds SSBNm with a Kd of 313 nM. Genotoxic stress analysis demonstrated that NmΔdinG was more sensitive to double-strand DNA breaks (DSB) induced by mitomycin C (MMC) than the Nm wildtype, defining the role of neisserial DinG in DSB repair. Notably, when NmΔdinG cells grown under MMC stress assessed by quantitative mass spectrometry, 134 proteins were shown to be differentially abundant (DA) compared to unstressed NmΔdinG cells. Among the DNA replication, repair and recombination proteins affected, polymerase III subunits and recombinational repair proteins RuvA, RuvB, RecB and RecD were significantly down regulated while TopA and SSB were upregulated under stress condition. Most of the other DA proteins detected are involved in metabolic functions. The present study shows that the helicase DinG is probably involved in regulating metabolic pathways as well as in genome maintenance.

Increasing evidence demonstrates that lysine acetylation is involved in Mycobacterium tuberculosis (Mtb) virulence and pathogenesis. However, previous investigations in Mtb have only monitored acetylation at lysine residues using selected reference strains. We analyzed the global N ε - and O-acetylation of three Mtb isolates: two lineage 7 clinical isolates and the lineage 4 H37Rv reference strain. Quantitative acetylome analysis resulted in identification of 2490 class-I acetylation sites, 2349 O-acetylation and 141 N ε -acetylation sites, derived from 953 unique proteins. Mtb O-acetylation was thereby significantly more abundant than N ε -acetylation. The acetylated proteins were found to be involved in central metabolism, translation, stress responses, and antimicrobial drug resistance. Notably, 261 acetylation sites on 165 proteins were differentially regulated between lineage 7 and lineage 4 strains. A total of 257 acetylation sites on 161 proteins were hypoacetylated in lineage 7 strains. These proteins are involved in Mtb growth, virulence, bioenergetics, host-pathogen interactions, and stress responses. This study provides the first global analysis of O-acetylated proteins in Mtb. This quantitative acetylome data expand the current understanding regarding the nature and diversity of acetylated proteins in Mtb and open a new avenue of research for exploring the role of protein acetylation in Mtb physiology.

DNA processing chain A (DprA) is a DNA-binding protein that is ubiquitous in bacteria and expressed in some archaea. DprA is active in many bacterial species that are competent for transformation of DNA, but its role in Neisseriameningitidis (Nm) is not well characterized. An Nm mutant lacking DprA was constructed, and the phenotypes of the wild-type and ΔdprA mutant were compared. The salient feature of the phenotype of dprA null cells is the total lack of competence for genetic transformation shown by all of the donor DNA substrates tested in this study. Here, Nm wild-type and dprA null cells appeared to be equally resistant to genotoxic stress. The gene encoding DprANm was cloned and overexpressed, and the biological activities of DprANm were further investigated. DprANm binds ssDNA more strongly than dsDNA, but lacks DNA uptake sequence-specific DNA binding. DprANm dimerization and interaction with the C-terminal part of the single-stranded binding protein SSBNmwere demonstrated. dprA is co-expressed with smg, a downstream gene of unknown function, and the gene encoding topoisomerase 1, topA.

In order to decipher the nature of the slowly growing Mycobacterium tuberculosis (M .tuberculosis) lineage 7, the differentially abundant proteins in strains of M. tuberculosis lineage 7 and lineage 4 were defined. Comparative proteomic analysis by mass spectrometry was employed to identify, quantitate and compare the protein profiles of strains from the two M. tuberculosis lineages. Label-free peptide quantification of whole cells from M. tuberculosis lineage 7 and 4 yielded the identification of 2825 and 2541 proteins, respectively. A combined total of 2867 protein groups covering 71% of the predicted M. tuberculosis proteome were identified. The abundance of 125 proteins in M. tuberculosis lineage 7 and 4 strains was significantly altered. Notably, the analysis showed that a number of M. tuberculosis proteins involved in growth and virulence were less abundant in lineage 7 strains compared to lineage 4. Five ABC transporter proteins, three phosphate binding proteins essential for inorganic phosphate uptake, and six components of the type 7 secretion system ESX-3 involved in iron acquisition were less abundant in M. tuberculosis lineage 7. This proteogenomic analysis provided an insight into the lineage 7-specific protein profile which may provide clues to understanding the differential properties of lineage 7 strains in terms of slow growth, survival fitness, and pathogenesis.

Within 1 week in April 2013, three cases of pulmonary tuberculosis (TB) were reported among students attending training sessions at an educational institution in Oslo, Norway. By the end of October 2013, a total of nine epidemiologically linked cases had been reported. The outbreak encompassed a total of 24 cases from 2009 to 2014, among which all of the 22 Mycobacterium tuberculosis isolates available had identical mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) profiles (MtbC15-9 code 10287-189) belonging to the Beijing lineage. Whole-genome sequencing (WGS) of the M. tuberculosis isolates revealed 20 variable nucleotide positions within the cluster, indicating a clonal outbreak. The most likely index case was identified and diagnosed in Norway in 2009 but was born in Asia. WGS analyses verified that all of the cases were indeed part of a single transmission chain. However, even when combining WGS and intensified contact tracing, we were unable to fully reconstruct the TB transmission events., (Copyright © 2017 American Society for Microbiology.)

Mitochondrial dysfunction and DNA damage is intimately connected to ageing and neurodegeneration, including Alzheimer's disease (AD). A particular culprit in this context is oxidative stress, which is a result of increased reactive oxygen species (ROS) due to hyperactive or dysfunctional mitochondria and/or reduced DNA repair capacity. Base excision repair (BER) is the major pathway for repairing oxidative damage events in chromosomal and mitochondrial DNA. Defects in BER have been detected in ageing and neurodegeneration. Mitochondrial transcription factor A (TFAM) plays an important role in the maintenance of mitochondrial DNA integrity. The present study investigated single nucleotide polymorphisms (SNPs) in the genes encoding the BER components MutYH, OGG1, APE1, PolB and PolG and the gene encoding mitochondrial TFAM in a cohort of 161 AD patients, 96 non-AD patient controls (PC) and 192 healthy controls (HC). Notably, the minor allele carriers of APE1 rs1130409 and the common allele carriers of TFAM rs1937 were associated with reduced mini-mental state examination score in AD patients, PC and HC, with no distinction of SNP frequencies in either of these sub-groups. Collectively, the results suggest an association between DNA maintenance and decline in cognitive function. These studies enlighten the normal brain aging process and point to potential new biomarkers for cognitive function and impairment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm. A transient reduction in transformation of DNA was observed in the Nm ΔrecG strain as compared to the wildtype. The gene encoding recGNm also contained an unusually high number of the DNA uptake sequence (DUS) that facilitate transformation in neisserial species. The differentially abundant protein profiles of the Nm wildtype and ΔrecG strains suggest that expression of RecGNm might be linked to expression of other proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity. This might explain the growth defect that was observed in the Nm ΔrecG null mutant., Competing Interests: The authors have declared that no competing interests exist.

Background: As an intracellular human pathogen, Mycobacterium tuberculosis (Mtb) is facing multiple stressful stimuli inside the macrophage and the granuloma. Understanding Mtb responses to stress is essential to identify new virulence factors and pathways that play a role in the survival of the tubercle bacillus. The main goal of this study was to map the regulatory networks of differentially expressed (DE) transcripts in Mtb upon various forms of genotoxic stress. We exposed Mtb cells to oxidative (H 2 O 2 or paraquat), nitrosative (DETA/NO), or alkylation (MNNG) stress or mitomycin C, inducing double-strand breaks in the DNA. Total RNA was isolated from treated and untreated cells and subjected to high-throughput deep sequencing. The data generated was analysed to identify DE genes encoding mRNAs, non-coding RNAs (ncRNAs), and the genes potentially targeted by ncRNAs., Results: The most significant transcriptomic alteration with more than 700 DE genes was seen under nitrosative stress. In addition to genes that belong to the replication, recombination and repair (3R) group, mainly found under mitomycin C stress, we identified DE genes important for bacterial virulence and survival, such as genes of the type VII secretion system (T7SS) and the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family. By predicting the structures of hypothetical proteins (HPs) encoded by DE genes, we found that some of these HPs might be involved in mycobacterial genome maintenance. We also applied a state-of-the-art method to predict potential target genes of the identified ncRNAs and found that some of these could regulate several genes that might be directly involved in the response to genotoxic stress., Conclusions: Our study reflects the complexity of the response of Mtb in handling genotoxic stress. In addition to genes involved in genome maintenance, other potential key players, such as the members of the T7SS and PE/PPE gene family, were identified. This plethora of responses is detected not only at the level of DE genes encoding mRNAs but also at the level of ncRNAs and their potential targets.

Background: Among children diagnosed to have chronic rheumatic valvular heart disease (RHD) in Ethiopia, many have been observed to develop recurrence of rheumatic fever (RF) despite secondary prophylaxis. This study determined the throat culture positivity rate and drug susceptibility pattern of beta hemolytic streptococci (BHS) isolated from children attending a specialized cardiac clinic in Ethiopia., Methods: Throat swabs were collected from 233 children receiving benzathine penicillin injection as secondary prophylaxis for RHD and cultured. The bacterial isolates were characterized using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Drug susceptibility was tested with the Kirby Bauer disc diffusion method. Anti-streptolysin O (ASO) titers were determined using ASO latex reagents., Results: The throat culture positivity rate for BHS was 24 % (56/233). Among the BHS bacterial strains isolated, four were characterized as S. pyogenes and another four as S. dysgalactiae subsp. equisimilis (Lancefield group A, C and G). All BHS were susceptible to penicillin except one isolate of S. agalactiae. Among 233 children enrolled, 46(19.7 %) showed increased ASO titer. Children who received antibiotic prophylaxis within 2-weeks of last injection had significantly lower BHS throat culture positivity rate than those injected every 4-weeks (p = 0.02). Children who missed at least one prophylaxis within the last 6 months had a higher BHS culture positivity rate than those who did not miss any (p = 0.0003)., Conclusions: The presence of groups A, C and G streptococci in the throat of children under secondary prophylaxis for RHD and increased ASO titer suggests failure of the regimen. This calls for further investigation into the causes of inadequate prophylaxis (including bioavailability of drugs used, optimal duration and patient compliance) and intervention.

Background: A deeply rooted phylogenetic lineage of Mycobacterium tuberculosis (M. tuberculosis) termed lineage 7 was discovered in Ethiopia. Whole genome sequencing of 30 lineage 7 strains from patients in Ethiopia was performed. Intra-lineage genome variation was defined and unique characteristics identified with a focus on genes involved in DNA repair, recombination and replication (3R genes)., Results: More than 800 mutations specific to M. tuberculosis lineage 7 strains were identified. The proportion of non-synonymous single nucleotide polymorphisms (nsSNPs) in 3R genes was higher after the recent expansion of M. tuberculosis lineage 7 strain started. The proportion of nsSNPs in genes involved in inorganic ion transport and metabolism was significantly higher before the expansion began. A total of 22346 bp deletions were observed. Lineage 7 strains also exhibited a high number of mutations in genes involved in carbohydrate transport and metabolism, transcription, energy production and conversion., Conclusions: We have identified unique genomic signatures of the lineage 7 strains. The high frequency of nsSNP in 3R genes after the phylogenetic expansion may have contributed to recent variability and adaptation. The abundance of mutations in genes involved in inorganic ion transport and metabolism before the expansion period may indicate an adaptive response of lineage 7 strains to enable survival, potentially under environmental stress exposure. As lineage 7 strains originally were phylogenetically deeply rooted, this may indicate fundamental adaptive genomic pathways affecting the fitness of M. tuberculosis as a species.

Background: Alzheimer's disease (AD) is a progressive, multifactorial neurodegenerative disorder that is the main cause of dementia globally. AD is associated with increased oxidative stress, resulting from imbalance in production and clearance of reactive oxygen species (ROS). ROS can damage DNA and other macromolecules, leading to genome instability and disrupted cellular functions. Base excision repair (BER) plays a major role in repairing oxidative DNA lesions. Here, we compared the expression of BER components APE1, OGG1, PARP1 and Polβ in blood and postmortem brain tissue from patients with AD, mild cognitive impairment (MCI) and healthy controls (HC)., Results: BER mRNA levels were correlated to clinical signs and cerebrospinal fluid biomarkers for AD. Notably, the expression of BER genes was higher in brain tissue than in blood samples. Polβ mRNA and protein levels were significantly higher in the cerebellum than in the other brain regions, more so in AD patients than in HC. Blood mRNA levels of OGG1 was low and PARP1 high in MCI and AD., Conclusions: These findings reflect the oxidative stress-generating energy-consumption in the brain and the importance of BER in repairing these damage events. The data suggest that alteration in BER gene expression is an event preceding AD. The results link DNA repair in brain and blood to the etiology of AD at the molecular level and can potentially serve in establishing novel biomarkers, particularly in the AD prodromal phase.

Expression of type IV pili (Tfp), filamentous appendages emanating from the bacterial surface, is indispensable for efficient neisserial transformation. Tfp pass through the secretin pore consisting of the membrane protein PilQ. PilG is a polytopic membrane protein, conserved in Gram-positive and Gram-negative bacteria, that is required for the biogenesis of neisserial Tfp. PilG null mutants are devoid of pili and non-competent for transformation. Here, recombinant full-length, truncated and mutated variants of meningococcal PilG were overexpressed, purified and characterized. We report that meningococcal PilG directly binds DNA in vitro, detected by both an electromobility shift analysis and a solid phase overlay assay. PilG DNA binding activity was independent of the presence of the consensus DNA uptake sequence. PilG-mediated DNA binding affinity was mapped to the N-terminus and was inactivated by mutation of residues 43 to 45. Notably, reduced meningococcal transformation of DNA in vivo was observed when PilG residues 43 to 45 were substituted by alanine in situ, defining a biologically significant DNA binding domain. N-terminal PilG also interacted with the N-terminal region of PilQ, which previously was shown to bind DNA. Collectively, these data suggest that PilG and PilQ in concert bind DNA during Tfp-mediated transformation.

Formation of a bacterial brain abscess entails loss of brain cells and formation of pus. The mechanisms behind the cell loss are not fully understood. Staphylococcus aureus, a common cause of brain abscesses, produces various exotoxins, including α-hemolysin, which is an important factor in brain abscess formation. α-Hemolysin may cause cytolysis by forming pores in the plasma membrane of various eukaryotic cells. However, whether α-hemolysin causes lysis of brain cells is not known. Nor is it known whether α-hemolysin in the brain causes cell death through pore formation or by acting as a chemoattractant, recruiting leukocytes and causing inflammation. Here we show that α-hemolysin injected into rat brain causes cell damage and edema formation within 30 min. Cell damage was accompanied by an increase in extracellular concentrations of zinc, GABA, glutamate, and other amino acids, indicating plasma membrane damage, but leukocytic infiltration was not seen 0.5-12h after α-hemolysin injection. This was in contrast to injection of S. aureus, which triggered extensive infiltration with neutrophils within 8h. In vitro, α-hemolysin caused concentration-dependent lysis of isolated nerve endings and cultured astrocytes. We conclude that α-hemolysin contributes to the cell death inherent in staphylococcal brain abscess formation as a pore-forming neurotoxin., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Recent genotyping studies of Mycobacterium tuberculosis in Ethiopia have reported the identification of a new phylogenetically distinct M. tuberculosis lineage, lineage 7. We therefore investigated the genetic diversity and association of specific M. tuberculosis lineages with sociodemographic and clinical parameters among pulmonary TB patients in the Amhara Region, Ethiopia. DNA was isolated from M. tuberculosis-positive sputum specimens (n=240) and analyzed by PCR and 24-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) analysis and spoligotyping. Bioinformatic analysis assigned the M. tuberculosis genotypes to global lineages, and associations between patient characteristics and genotype were evaluated using logistic regression analysis. The study revealed a high diversity of modern and premodern M. tuberculosis lineages, among which approximately 25% were not previously reported. Among the M. tuberculosis strains (n=138) assigned to seven subgroups, the largest cluster belonged to the lineage Central Asian (CAS) (n=60; 26.0%), the second largest to lineage 7 (n=36; 15.6%), and the third largest to the lineage Haarlem (n=35; 15.2%). Four sublineages were new in the MIRU-VNTRplus database, designated NW-ETH3, NW-ETH1, NW-ETH2, and NW-ETH4, which included 24 (10.4%), 18 (7.8%), 8 (3.5%), and 5 (2.2%) isolates, respectively. Notably, patient delay in seeking treatment was significantly longer among patients infected with lineage 7 strains (Mann-Whitney test, P<0.008) than in patients infected with CAS strains (adjusted odds ratio [AOR], 4.7; 95% confidence interval [CI], 1.6 to 13.5). Lineage 7 strains also grew more slowly than other M. tuberculosis strains. Cases of Haarlem (OR, 2.8; 95% CI, 1.2 to 6.6) and NW-ETH3 (OR, 2.8; 95% CI, 1.0 to 7.3) infection appeared in defined clusters. Intensified active case finding and contact tracing activities in the study region are needed to expedite diagnosis and treatment of TB., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)