Molecular insight into thymoquinone mechanism of action against Mycobacterium tuberculosis.

Natural products are promising antimicrobials, usually having multiple and different cellular targets than synthetic antibiotics. Their influence on bacteria at various metabolic and functional levels contributes to higher efficacy even against drug-resistant strains. One such compound is a naturally occurring p-benzoquinone - thymoquinone. It is effective against different bacteria, including multidrug-resistant and extremely drug-resistant Mycobacterium tuberculosis. Its antibacterial mechanism of action was studied in several bacterial species except mycobacteria. To get an insight into the antimycobacterial activity of thymoquinone at the molecular level, we performed metabolomic and transcriptomic analyzes of bacteria exposed to this compound. The expression of genes coding stress-responsive sigma factors revealed that thymoquinone rapidly induces the production of sigE transcripts. At the same time, prolonged influence results in the overexpression of all sigma factor genes and significantly upregulates sigF. The metabolomic analysis confirmed that the antimycobacterial activity of thymoquinone was related to the depletion of NAD and ATP pools and the downregulation of plasma membrane lipids. This state was observed after 24 h and was persistent the next day, suggesting that bacteria could not activate catabolic mechanisms and produce energy. Additionally, the presence of a thymoquinone nitrogen derivative in the bacterial broth and the culture was reported.

Sulphides from garlic essential oil dose-dependently change the distribution of glycerophospholipids and induce N6-tuberculosinyladenosine formation in mycobacterial cells.

The antimicrobial properties of garlic are widely known, and numerous studies confirmed its ability to inhibit the growth of Mycobacterium tuberculosis. In this work, we explored the molecular mechanism of action of sulphides present in garlic essential oil against mycobacteria. The targeted transcriptomics and untargeted LC-MS metabolomics were applied to study dose- and time-dependent metabolic changes in bacterial cells under the influence of stressing agent. Expression profiles of genes coding stress-responsive sigma factors regulatory network and metabolic observations proved that sulphides from garlic essential oil are an efficient and specific agent affecting glycerophospholipids levels and their distribution within the cell envelope. Additionally, sulphides induced the Dimroth rearrangement of 1-Tuberculosinyladenosine to N6-tuberculosinyladenosine in mycobacterial cells as a possible neutralization mechanism protecting the cell from a basic nucleophilic environment. Sulphides affected cell envelope lipids and formation of N6-tuberculosinyladenosine in M. tuberculosis.

Exposure to Nepalese Propolis Alters the Metabolic State of Mycobacterium tuberculosis.

Propolis is a natural product proved to be efficient against Mycobacterium tuberculosis. Although it is produced by bees, its active alcoholic-aqueous fraction contains plant-derived molecules. To gain some insight into its mechanism of antimycobacterial activity, we studied the metabolic changes in bacterial cells treated with extract of Trigona sp. propolis from Nepal. The detailed metabolomic and transcriptomic analysis performed in this study indicated target points in bacterial cells under propolis extract influence. The profile of lipids forming the outer and middle layer of the mycobacterial cell envelope was not changed by propolis treatment, however, fluctuations in the profiles of amphipathic glycerophospholipids were observed. The enrichment analysis revealed bacterial metabolic pathways affected by Trigona sp. propolis treatment. The early metabolic response involved much more pathways than observed after 48 h of incubation, however, the highest enrichment ratio was observed after 48 h, indicating the long-lasting influence of propolis. The early bacterial response was related to the increased demand for energy and upregulation of molecules involved in the formation of the cell membrane. The transcriptomic analysis confirmed that bacteria also suffered from oxidative stress, which was more pronounced on the second day of exposure. This was the first attempt to explain the action of Nepalese propolis extract against mycobacteria.

Investigating the Antituberculosis Activity of Selected Commercial Essential Oils and Identification of Active Constituents Using a Biochemometrics Approach and In Silico Modeling.

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis which has become prevalent due to the emergence of resistant M. tuberculosis strains. The use of essential oils (EOs) as potential anti-infective agents to treat microbial infections, including TB, offers promise due to their long historical use and low adverse effects. The current study aimed to investigate the in vitro anti-TB activity of 85 commercial EOs, and identify compounds responsible for the activity, using a biochemometrics approach. A microdilution assay was used to determine the antimycobacterial activity of the EOs towards some non-pathogenic Mycobacterium strains. In parallel, an Alamar blue assay was used to investigate antimycobacterial activity towards the pathogenic M. tuberculosis strain. Chemical profiling of the EOs was performed using gas chromatography-mass spectrometry (GC-MS) analysis. Biochemometrics filtered out putative biomarkers using orthogonal projections to latent structures discriminant analysis (OPLS-DA). In silico modeling was performed to identify potential therapeutic targets of the active biomarkers. Broad-spectrum antimycobacterial activity was observed for Cinnamomum zeylanicum (bark) (MICs = 1.00, 0.50, 0.25 and 0.008 mg/mL) and Levisticum officinale (MICs = 0.50, 0.5, 0.5 and 0.004 mg/mL) towards M. smegmatis, M. fortuitum, M. gordonae and M. tuberculosis, respectively. Biochemometrics predicted cinnamaldehyde, thymol and eugenol as putative biomarkers. Molecular docking demonstrated that cinnamaldehyde could serve as a scaffold for developing a novel class of antimicrobial compounds by targeting FtsZ and PknB from M. tuberculosis.

Unveiling the Phytochemical Profile and Biological Potential of Five Artemisia Species.

The Artemisia L. genus comprises over 500 species with important medicinal and economic attributes. Our study aimed at providing a comprehensive metabolite profiling and bioactivity assessment of five Artemisia species collected from northeastern Romania (A. absinthium L., A. annua L., A. austriaca Jacq., A. pontica L. and A. vulgaris L.). Liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS/MS) analysis of methanol and chloroform extracts obtained from the roots and aerial parts of the plants led to the identification of 15 phenolic acids (mostly hydroxycinnamic acid derivatives), 26 flavonoids (poly-hydroxylated/poly-methoxylated flavone derivatives, present only in the aerial parts), 14 sesquiterpene lactones, 3 coumarins, 1 lignan and 7 fatty acids. Clustered image map (CIM) analysis of the phytochemical profiles revealed that A. annua was similar to A. absinthium and that A. pontica was similar to A. austriaca, whereas A. vulgaris represented a cluster of its own. Correlated with their total phenolic contents, the methanol extracts from both parts of the plants showed the highest antioxidant effects, as assessed by the DPPH and ABTS radical scavenging, CUPRAC, FRAP and total antioxidant capacity methods. Artemisia extracts proved to be promising sources of enzyme inhibitory agents, with the methanol aerial part extracts being the most active samples against acetylcholinesterase and glucosidase. All Artemisia samples displayed good antibacterial effects against Mycobacterium tuberculosis H37Ra, with MIC values of 64-256 mg/L. In conclusion, the investigated Artemisia species proved to be rich sources of bioactives endowed with antioxidant, enzyme inhibitory and anti-mycobacterial properties.

The Landscape of Gene Expression during Hyperfilamentous Biofilm Development in Oral Candida albicans Isolated from a Lung Cancer Patient.

The filamentation ability of Candida albicans represents one of the main virulence factors allowing for host tissue penetration and biofilm formation. The aim of this paper was to study the genetic background of the hyperfilamentous biofilm development in vitro in C. albicans isolated from the oral cavity of a lung cancer patient. Analyzed C. albicans isolates (CA1, CA2, CA3) were chosen based on their different structures of mature biofilm. The CA3 isolate possessing hyperfilamentation properties and forming high biofilm was compared with CA1 and CA2 isolates exhibiting low or average biofilm-forming ability, respectively. The detailed biofilm organization was studied with the use of confocal scanning laser microscopy. The whole transcriptome analysis was conducted during three stages of biofilm development (24 h, 48 h, 72 h). In contrast to CA1 and/or CA2 isolate, the CA3 isolate was characterized by a significant upregulation of genes encoding for cell wall proteins (HWP1, PGA13, PGA44, ALS3) and candidalysin (ECE1), as well as being involved in iron metabolism (FRE1, ALS3), sulfur metabolism (HAL21), the degradation of aromatic compounds (HQD2), and membrane transport (DIP5, PHO89, TNA1). In contrast, some genes (SCW11, FGR41, RBE1) in the CA3 were downregulated. We also observed the overexpression of a few genes over time-mainly FRE1, ATX1, CSA2 involved in iron metabolism. This is the first insight into the potential function of multiple genes in the hyperfilamentous biofilm formation in C. albicans, primarily isolated from host tissue, which may have an important clinical impact on cancer patients. Moreover, the presented data can lay the foundation for further research on novel pathogen-specific targets for antifungal drugs.

Tanshinones from Salvia miltiorrhiza inhibit Mycobacterium tuberculosis via disruption of the cell envelope surface and oxidative stress.

The unique structure of Mycobacterium tuberculosis cell envelope provides impermeable barrier against environmental stimuli. In the situation that this barrier is disturbed Mycobacteria react at the transcriptional and translational level to redirect metabolic processes and to maintain integrity of the cell. In this work we aimed to explore the early metabolic response of M. tuberculosis to tanshinones, which are active antimycobacterial compounds of Salvia miltiorrhiza Bunge root. The investigation of the expression of sigma factors revealed the significant shifts in the general bacterial regulatory network, whereas LC-MS metabolomics evidenced the changes in the composition of bacterial cell envelope and indicated altered metabolic pathways. Tanshinones acted via the disruption of the cell envelope surface and generation of reactive oxygen species. Bacteria responded with overproduction of inner region of outer membrane, fluctuations in the production of glycerophosphoinositolglycans, as well as changes in the levels of mycobactins, accompanied by enrichment of metabolic pathways related to redox balance and repair of damages caused by tanshinones.

Usnic Acid Treatment Changes the Composition of Mycobacterium tuberculosis Cell Envelope and Alters Bacterial Redox Status.

Mycobacterium tuberculosis developed efficient adaptation mechanisms in response to different environmental conditions. This resulted in the ability to survive in human macrophages and in resistance to numerous antibiotics. To get insight into bacterial responses to potent antimycobacterial natural compounds, we tested how usnic acid, a lichen-derived secondary metabolite, would influence mycobacteria at transcriptomic and metabolomic levels. The analysis of expression of sigma factors revealed a profound impact of usnic acid on one of the primary genetic regulatory systems of M. tuberculosis Combined liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses allowed us to observe the perturbations in metabolic pathways, as well as in lipid composition, which took place within 24 h of exposure. Early bacterial response was related to redox homeostasis, lipid synthesis, and nucleic acid repair. Usnic acid treatment provoked disturbances of redox state in mycobacterial cells and increased production of structural elements of the cell wall and cell membrane. In addition, to increase the number of molecules related to restoration of redox balance, the rearrangements of the cell envelope were the first defense mechanisms observed under usnic acid treatment.IMPORTANCE The evaluation of mechanisms of mycobacterial response to natural products has been barely studied. However, it might be helpful to reveal bacterial adaptation strategies, which are eventually crucial for the discovery of new drug targets and, hence, understanding the resistance mechanisms. This study showed that the first-line mycobacterial defense against usnic acid, a potent antimicrobial agent, is the remodeling of the cell envelope and restoring redox homeostasis. Transcriptomic data correlated with metabolomics analysis. The observed metabolic changes appeared similar to those exerted by antibiotics.

Ocular Involvement of SARS-CoV-2 in a Polish Cohort of COVID-19-Positive Patients.

The coronavirus SARS-CoV-2 responsible for the current human COVID-19 pandemic has shown tropism toward different organs with variable efficiency, eyes included. The purpose of this study has been to investigate the presence of detectable SARS-CoV-2 infection in ocular swabs in patients affected by COVID-19. A consecutive series of 74 COVID-19-positive patients (age 21-89) were enrolled at two Polish COVID-19 hospitals for 4 months and were characterized by PCR for the presence of the SARS-CoV-2 genetic material in nasopharyngeal (NP) and ocular swabs, while their respiratory and ocular symptoms were noted. Almost 50% of them presented with severe/critical respiratory involvement, and some degree of eye disease. No tight correlation was observed between the presence of ocular and respiratory symptoms. Three male patients presenting with severe/critical lung disease tested positive in ocular swab, however with mild/moderate ocular symptoms. In conclusion, our study lends further support to the view that overt ocular infection by the SARS-CoV-2 virus is not such a frequent occurrence.

Sample pooling as a strategy for community monitoring for SARS-CoV-2.

Sample pooling strategy was intended to determine the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2 and process them without significant loss of test usability. Standard molecular diagnostic laboratory equipment, and commercially available centrifugal filters, RNA isolation kits and SARS Cov2 PCR tests were used. The basic idea was to combine and concentrate several samples to the maximal volume, which can be extracted with the single extraction column. Out of 16 tested pools, 12 were positive with cycle threshold (Ct) values within 0.5 and 3.01 Ct of the original individual specimens. The analysis of 112 specimens determined that 12 pools were positive, followed by identification of 6 positive individual specimens among the 112 tested. This testing was accomplished with the use of 16 extractions/PCR tests, resulting in saving of 96 reactions but adding the 40 centrifugal filters. The present study demonstrated that pool testing could detect even up to a single positive sample with Ct value as high as 34. According to the standard protocols, reagents and equipment, this pooling method can be applied easily in current clinical testing laboratories.

Untargetted Metabolomic Exploration of the Mycobacterium tuberculosis Stress Response to Cinnamon Essential Oil.

The antimycobacterial activity of cinnamaldehyde has already been proven for laboratory strains and for clinical isolates. What is more, cinnamaldehyde was shown to threaten the mycobacterial plasma membrane integrity and to activate the stress response system. Following promising applications of metabolomics in drug discovery and development we aimed to explore the mycobacteria response to cinnamaldehyde within cinnamon essential oil treatment by untargeted liquid chromatography-mass spectrometry. The use of predictive metabolite pathway analysis and description of produced lipids enabled the evaluation of the stress symptoms shown by bacteria. This study suggests that bacteria exposed to cinnamaldehyde could reorganize their outer membrane as a physical barrier against stress factors. They probably lowered cell wall permeability and inner membrane fluidity, and possibly redirected carbon flow to store energy in triacylglycerols. Being a reactive compound, cinnamaldehyde may also contribute to disturbances in bacteria redox homeostasis and detoxification mechanisms.

Mycobacterium tuberculosis topoisomerases and EthR as the targets for new anti-TB drugs development.

The significant increase in the detection of drug-resistant strains of Mycobacterium tuberculosis caused an urgent need for the discovery new antituberculosis drugs. Development of bioinformatics and computational sciences enabled the progress of new strategies leading to design, discovery and identification of a series of interesting drug candidates. In this short review, we would like to present recently discovered compounds targeting important mycobacterial proteins: DNA topoisomerases and the transcriptional repressor of EthA monooxygenase - EthR.

Antimycobacterial Activity of Cinnamaldehyde in a Mycobacterium tuberculosis(H37Ra) Model.

The purpose of the study was to evaluate the antimycobacterial activity and the possible action mode of cinnamon bark essential oil and its main constituent-cinnamaldehyde-against the Mycobacterium tuberculosis ATCC 25177 strain. Cinnamaldehyde was proved to be the main bioactive compound responsible for mycobacterial growth inhibition and bactericidal effects. The antimycobacterial activity of cinnamaldehyde was found to be comparable with that of ethambutol, one of the first-line anti-TB antibiotics. The selectivity index determined using cell culture studies in vitro showed a high biological potential of cinnamaldehyde. In M. tuberculosis cells exposed to cinnamaldehyde the cell membrane stress sensing and envelope preserving system are activated. Overexpression of clgR gene indicates a threat to the stability of the cell membrane and suggests a possible mechanism of action. No synergism was detected with the basic set of antibiotics used in tuberculosis treatment: ethambutol, isoniazid, streptomycin, rifampicin, and ciprofloxacin.

Nigella damascena L. Essential Oil-A Valuable Source of ß-Elemene for Antimicrobial Testing.

The most commonly used plant source of ß-elemene is Curcuma wenyujin Y. H. Chen & C. Ling (syn. of Curcuma aromatic Salisb.) with its content in supercritical CO2 extract up to 27.83%. However, the other rich source of this compound is Nigella damascena L. essential oil, in which ß-elemene accounts for 47%. In this work, the effective protocol for preparative isolation of ß-elemene from a new source-N. damascena essential oil-using high performance counter-current chromatography HPCCC was elaborated. Furthermore, since sesquiterpens are known as potent antimicrobials, the need for finding new agents designed to combat multi-drug resistant strains was addressed and the purified target compound and the essential oil were tested for its activity against a panel of Gram-positive and Gram-negative bacteria, fungi, and mycobacterial strains. The application of the mixture of petroleum ether, acetonitrile, and acetone in the ratio 2:1.5:0.5 (v/v) in the reversed phase mode yielded ß-elemene with high purity in 70 min. The results obtained for antimicrobial assay clearly indicated that N. damascena essential oil and isolated ß-elemene exert action against Mycobacterium tuberculosis strain H37Ra.

The Effect of Combining Natural Terpenes and Antituberculous Agents against Reference and Clinical Mycobacterium tuberculosis Strains.

Background: On account of emergence of multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains, combinations of drugs with natural compounds were tested to search for antibiotic activity enhancers. In this work we studied terpenes (α-pinene, bisabolol, ß-elemene, (R)-limonene, (S)-limonene, myrcene, sabinene), which are the main constituents of essential oil obtained from Mutellina purpurea L., a plant with described antitubercular activity, to investigate their interactions with antibiotics against reference Mtb strains and multidrug-resistant clinical isolates. Methods: The serial dilution method was used to evaluate the minimal inhibitory concentration (MIC) of tested compounds, while the fractional inhibitory concentration index (FICI) was calculated for characterization of interactions. Moreover, IC50 values of tested compounds were determined using monkey kidney epithelial cell line (GMK). Results: The combinations of all studied terpenes with ethambutol or rifampicin resulted in a synergistic interaction. Bisabolol and (R)-limonene decreased the MIC for rifampicin at least two-fold for all tested strains, however no synergistic action was observed against virulent strains. The tested terpenes showed slight (bisabolol) or no cytotoxic effect against normal eukaryotic cells in vitro. Conclusions: The obtained enhanced activity (FICI < 0.5) of ethambutol and rifampicin against H37Ra strain under the influence of the studied terpenes may be correlated to the capability of essential oil constituents to modify bacterial resistance mechanisms in general. The observed differences in avirulent and virulent bacteria susceptibility to terpenes tested separately and in combinations with antibiotics can be correlated with the differences in the cell wall structure between H37Ra mutant and all virulent strains.

The frequently occurring components of essential oils beta elemene and R-limonene alter expression of dprE1 and clgR genes of Mycobacterium tuberculosis H37Ra.

In the past few years, there has been a significant increase in detection of drug resistant strains of Mycobacterium tuberculosis. Search for new antimycobacterial drugs brought natural sources with their chemical diversity in focus. Especially essential oils, produced by plants also for toxic effect, are reservoir of potentially antitubercular compounds. In the present work, we exposed M. tuberculosis H37Ra ATCC 25177 strain to some terpenes commonly occurring in essential oils. Gene expression profiling was used to explore possible influence of these compounds on stress sensing and envelope preserving function. Expression of two genes dprE1 involved in cell wall synthesis and clgR responsible for regulation of cell membrane preservation was investigated. We report that two out of five tested compounds: ß-elemene and R-limonene alter expression of dprE1 and clgR genes. These findings indicate various mechanisms of action of essential oils compounds on M. tuberculosis. Especially the clgR expression seemed to be the perfect marker of stress sensing and envelope preserving systems status.

LC-QTOF-MS Analysis and Activity Profiles of Popular Antioxidant Dietary Supplements in Terms of Quality Control.

The dietary supplements with claimed antioxidant activity constitute a substantial part of the dietary supplement market. In this study, we performed the LC-QTOF-MS analysis and investigated the activity profiles of popular antioxidant dietary supplements from different chemical groups in terms of quality control. The commonly used antioxidant tests and statistical analysis revealed that substantial part of the results was comparable if 1 g sample was considered, but while comparing single and daily doses, significant differences in antioxidant values were noticed in all assays. The best antioxidant activity was obtained in ORAC assay (from 142 to 13814 µM of Trolox equivalents per 1 g of sample), and the strongest correlation occurred between TPC and ORAC. The LC-QTOF-MS analysis revealed that catechins were present in samples having the best antioxidant activity and that dietary supplements showing the weakest activity contained very small amount of any chemical constituents.

Natural Terpenes Influence the Activity of Antibiotics against Isolated Mycobacterium tuberculosis.

OBJECTIVE: In this study, we aimed to describe the influence of natural terpenes on the antimycobacterial activity of first-line tuberculostatic drugs against isolated Mycobacterium tuberculosis. MATERIALS AND METHODS: The natural terpenes used in this study were R-limonene, S-limonene, myrcene, sabinene, α-pinene, and ß-elemene. The values of the minimum inhibitory concentration (MIC) for these terpenes, as well as for combinations of terpenes with tuberculostatic antibiotics (ethambutol, isoniazid, and rifampicin), were determined using a tube log2 dilution method in the range of 125-0.059 µg/mL. RESULTS: S-limonene had a strong synergistic effect with all tested antibiotics (MIC decreased from 16 to 0.475 µg/mL for ethambutol, from 16 to 0.237 µg/mL for rifampicin, and from 32 to 0.475 µg/mL for isoniazid). Combinations of myrcene, R-limonene, ß-elemene, and sabinene with tuberculostatic antibiotics resulted in a decreased MIC of the antibiotics (from 3.9 to 0.475 µg/mL for ethambutol, from 15 to 0.475 µg/mL for isoniazid, and from 0.475 to 0.237 µg/mL for rifampicin) while combinations of α-pinene with ethambutol and isoniazid resulted in increased MIC values (from 16 to 125 µg/mL for ethambutol, and from 32 to 125 µg/mL for isoniazid). Rifampicin had a synergistic increase in activity with all the tested compounds. CONCLUSIONS: Our study showed that terpenes enhance the activity of tuberculostatic antibiotics.

Essential Oils of some Mentha Species and Cultivars, their Chemistry and Bacteriostatic Activity.

The major purpose of this study was to determine the chemical relationships between the essential oils (EO) obtained form 18 mint samples of different species and its cultivrs. GC/MS analysis of all essential oils showed that oxygenated monoterpenoids are the major components of these oils, with the exception of Mentha arvensis 'Banana'. Based on the. chemical composition of the essential oils, the analyzed mints can be divided into five groups. Group I was characterized by the-presence of menthol and menthone, piperitenone oxide is the major constituent of group II, linalool of group III, carvone characterizes group IV, while 3-octanone is the most characteristic compound for group V. A reference strain of biofilm forming Staphylococcus epidermidis ATTC 35984 was tested against the obtained essential oils. Two of them, M suaveolens 'Variegata' and M x piperita 'Almira' exhibited significant bacteriostatic activity. The MIC values of these EOs were 0.25% and 0.5% respectively in comparison with an average MIC of 4% of the other tested mint EOs. Both active EOs are characterized by the presence of piperitenone oxide as the major component.

Morphological Changes in the Overall Mycobacterium tuberculosis H37Ra Cell Shape and Cytoplasm Homogeneity due to Mutellina purpurea L. Essential Oil and Its Main Constituents.

OBJECTIVE: The aim of this study was to evaluate the antimycobacterial activity of the essential oil (EO) of Mutellina purpurea L. and its main constituents against the M. tuberculosis H37Ra strain. MATERIALS AND METHODS: The M. purpurea EO was obtained by hydrodistillation, while its main constituents were purchased. The minimal inhibitory concentration values were determined by the log2 dilution method. Visualization of the effects of the tested substances on M. tuberculosis was performed using a transmission electron microscope (TEM). Mathematical shape descriptors such as area, circularity, aspect ratio and roundness were calculated to describe morphological changes in bacterial cell shape. RESULTS: The EO of M.purpurea and all substances tested in this experiment showed a significant antimycobacterial activity. The most active was α-pinene followed by bisabolol and myrcene (8, 16 and 32 µg/ml, respectively). The EO and limonene exhibited the same antimicrobial activity (64 µg/ml). The TEM images and shape descriptors showed significant changes in the overall tuberculosis cell shape and cytoplasm homogeneity (uniformity and consistency) CONCLUSIONS: In this study, the low molecular weight compounds of mono- and sesquiterpenes penetrated/destabilized the complex mycobacterial cell wall and decreased its viability. There is a need for further experiments to explain the mechanism of action of these small particles.