Evaluation of In Vitro Synergistic Effects of Tetracycline with Alkaloid-Related Compounds against Diarrhoeic Bacteria.

Diarrhoea remains an important public health concern, particularly in developing countries, and has become difficult to treat because of antibacterial resistance. The development of synergistic antimicrobial agents appears to be a promising alternative treatment against diarrhoeic infections. In this study, the combined effect of tetracycline together with either nitroxoline, sanguinarine, or zinc pyrithione (representing various classes of plant-based compounds) was evaluated in vitro against selected diarrhoeic bacteria (Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Shigella flexneri, Vibrio parahaemolyticus, and Yersinia enterocolitica). The chequerboard method in 96-well microtiter plates was used to determine the sum of the fractional inhibitory concentration indices (FICIs). Three independent experiments were performed per combination, each in triplicate. It was observed that the combination of tetracycline with either nitroxoline, sanguinarine, or zinc pyrithione produced synergistic effects against most of the pathogenic bacteria tested, with FICI values ranging from 0.086 to 0.5. Tetracycline-nitroxoline combinations produced the greatest synergistic action against S. flexneri at a FICI value of 0.086. The combinations of the agents tested in this study can thus be used for the development of new anti-diarrhoeic medications. However, studies focusing on their in vivo anti-diarrhoeic activity and safety are required before any consideration for utilization in human medicine.

Insights into the Detoxification of Spruce Monoterpenes by the Eurasian Spruce Bark Beetle.

Plant defence mechanisms, including physical barriers like toughened bark and chemical defences like allelochemicals, are essential for protecting them against pests. Trees allocate non-structural carbohydrates (NSCs) to produce secondary metabolites like monoterpenes, which increase during biotic stress to fend off pests like the Eurasian spruce bark beetle, ESBB (Ips typographus). Despite these defences, the ESBB infests Norway spruce, causing significant ecological damage by exploiting weakened trees and using pheromones for aggregation. However, the mechanism of sensing and resistance towards host allelochemicals in ESBB is poorly understood. We hypothesised that the exposure of ESBB to spruce allelochemicals, especially monoterpenes, leads to an upsurge in the important detoxification genes like P450s, GSTs, UGTs, and transporters, and at the same time, genes responsible for development must be compromised. The current study demonstrates that exposure to monoterpenes like R-limonene and sabiene effectively elevated detoxification enzyme activities. The differential gene expression (DGE) analysis revealed 294 differentially expressed (DE) detoxification genes in response to R-limonene and 426 DE detoxification genes in response to sabiene treatments, with 209 common genes between the treatments. Amongst these, genes from the cytochrome P450 family 4 and 6 genes (CP4 and CP6), esterases, glutathione S-transferases family 1 (GSTT1), UDP-glucuronosyltransferase 2B genes (UDB), and glucose synthesis-related dehydrogenases were highly upregulated. We further validated 19 genes using RT-qPCR. Additionally, we observed similar high expression levels of detoxification genes across different monoterpene treatments, including myrcene and α-pinene, suggesting a conserved detoxification mechanism in ESBB, which demands further investigation. These findings highlight the potential for molecular target-based beetle management strategies targeting these key detoxification genes.

Susceptibility of Staphylococcus aureus to Anti-Inflammatory Drugs with a Focus on the Combinatory Effect of Celecoxib with Oxacillin In Vitro.

Musculoskeletal infections (MIs) are among the most difficult-to-treat staphylococcal diseases due to antibiotic resistance. This has encouraged the development of innovative strategies, such as combination therapy, to combat MI. The aim of this study was to investigate the in vitro antistaphylococcal activity of anti-inflammatory drugs and the combined antimicrobial effect of celecoxib and oxacillin. The minimum inhibitory concentrations (MICs) of 17 anti-inflammatory drugs against standard strains and clinical isolates of S. aureus, including methicillin-resistant strains (MRSAs), were determined using the broth microdilution method. The fractional inhibitory concentration indices (FICIs) were evaluated using checkerboard assays. Celecoxib produced the most potent antistaphylococcal effect against all tested strains (MICs ranging from 32 to 64 mg/L), followed by that of diacerein against MRSA3 and MRSA ATCC 33592 (MIC 64 mg/L). Several synergistic effects were observed against the tested S. aureus strains, including MRSA (FICI ranging from 0.087 to 0.471). The strongest synergistic interaction (FICI 0.087) was against MRSA ATCC 33592 at a celecoxib concentration of 2 mg/L, with a 19-fold oxacillin MIC reduction (from 512 to 26.888 mg/L). This is the first report on the combined antistaphylococcal effect of celecoxib and oxacillin. These findings suggest celecoxib and its combination with oxacillin as perspective agents for research focused on the development of novel therapies for MI caused by S. aureus. This study further indicates that celecoxib could resensitize certain MRSA strains, in some cases, to be susceptible to ß-lactams (e.g., oxacillin) that were not previously tested. It is essential to mention that the in vitro concentrations of anti-inflammatory drugs are higher than those typically obtained in patients. Therefore, an alternative option for its administration could be the use of a drug delivery system for the controlled slow release from an implant at the infection site.

Validation of Broth Macrodilution Volatilization Method for Testing of Essential Oils in Liquid and Vapor Phase: Chemical Composition, Cytotoxicity, and Antibacterial Effect of Indian Medicinal Plants against Pneumonia-Causing Pathogens.

Essential oils (EOs) have great potential in inhalation therapy for the treatment of respiratory infections. However, innovative methods for evaluation of antimicrobial activity of their vapors are still needed. The current study reports validation of the broth macrodilution volatilization method for assessment of the antibacterial properties of EOs and shows the growth-inhibitory effect of Indian medicinal plants against pneumonia-causing bacteria in liquid and vapor phase. Among all samples tested, Trachyspermum ammi EO exhibits the strongest antibacterial effect against Haemophilus influenzae, with minimum inhibitory concentrations of 128 and 256 µg/mL in the liquid and vapor phases, respectively. Furthermore, Cyperus scariosus EO is found to be nontoxic to normal lung fibroblasts assessed by modified thiazolyl blue tetrazolium bromide assay. Chemical analysis performed using gas chromatography-mass spectrometry identified α-citral, cyperotundone, and thymol as the main constituents of Cymbopogon citratus, C. scariosus, and T. ammi EOs, respectively. In addition, ß-cymene is identified as the major compound of T. ammi EO vapors when analyzed using solid-phase microextraction and gas-tight syringe sampling techniques. This study demonstrates the validity of the broth macrodilution volatilization method for antimicrobial screening of volatile compounds in the vapor phase and suggests the therapeutic potential of Indian medicinal plants in inhalation therapy.

Antibacterial Activity of Thymus vulgaris L. Essential Oil Vapours and Their GC/MS Analysis Using Solid-Phase Microextraction and Syringe Headspace Sampling Techniques.

While the inhalation of Thymus vulgaris L. essential oil (EO) is commonly approved for the treatment of mild respiratory infections, there is still a lack of data regarding the antimicrobial activity and chemical composition of its vapours. The antibacterial activity of the three T. vulgaris EOs against respiratory pathogens, including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes, was assessed in both liquid and vapour phases using the broth microdilution volatilisation (BMV) method. With the aim of optimising a protocol for the characterisation of EO vapours, their chemical profiles were determined using two headspace sampling techniques coupled with GC/MS: solid-phase microextraction (HS-SPME) and syringe headspace sampling technique (HS-GTS). All EO sample vapours exhibited antibacterial activity with minimum inhibitory concentrations (MIC) ranging from 512 to 1024 µg/mL. According to the sampling technique used, results showed a different distribution of volatile compounds. Notably, thymol was found in lower amounts in the headspace-peak percentage areas below 5.27% (HS-SPME) and 0.60% (HS-GTS)-than in EOs (max. 48.65%), suggesting that its antimicrobial effect is higher in vapour. Furthermore, both headspace sampling techniques were proved to be complementary for the analysis of EO vapours, whereas HS-SPME yielded more accurate qualitative results and HS-GTS proved a better technique for quantitative analysis.

New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro.

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (ß-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase.

Volatile Antimicrobial Agents and In Vitro Methods for Evaluating Their Activity in the Vapour Phase: A Review.

This review summarizes data on the in vitro antimicrobial effectiveness of volatile agents of plant origin and in vitro methods for evaluating their activity in the vapour phase. As a result of literature analysis, the antimicrobial efficacy of vapours from 122 different plant species and 19 pure compounds examined in 61 studies using different in vitro tests against a broad spectrum of microorganisms was identified and summarized. In addition, 11 different techniques found in the literature are described in detail. An original classification of methods based on the solid and liquid matrix volatilization principle is proposed because carrier medium/matrix selection is crucial for the volatilization of any agents tested. This review should be useful for medicinal, pharmaceutical, food, and agricultural experts working in areas related to the management of infectious diseases (especially respiratory and skin infections), food preservation (active packaging), and protection of agriculture products (controlled atmosphere). It may also stimulate the interest of pharmaceutical, cosmetic, food, and agriculture industries in the research and development of new antimicrobial agents of natural origin. Since several original apparatuses previously developed for antimicrobial susceptibility testing in the vapour phase are described in this review, labware manufacturers may also be interested in this topic. The review also provides specific guidelines and recommendations for researchers studying the antimicrobial activity of volatile agents. The article will therefore appeal to communities of industrial stakeholders, pharmacists, physicians, food experts, agriculturists, and researchers in related areas such as pharmacology, medicinal chemistry, microbiology, natural product chemistry, food preservation and plant protection.

Vapors of Volatile Plant-Derived Products Significantly Affect the Results of Antimicrobial, Antioxidative and Cytotoxicity Microplate-Based Assays.

Volatile plant-derived products were observed to exhibit broad spectrum of biological effects. However, due to their volatility, results of conventional microplate-based bioassays can be significantly affected by the vapors. With aim to demonstrate this phenomenon, antimicrobial, antioxidant, and cytotoxic activities of three essential oils (Alpinia elegans, Cinnamomum iners, and Xanthostemon verdugonianus), one supercritical CO2 extract (Nigella sativa), and four plant-derived compounds (capsaicin, caryophyllene oxide, 8-hydroxyquinoline, and thymoquinone) were evaluated in series of experiments including both ethylene vinyl acetate (EVA) Capmat sealed and nonsealed microplates. The results clearly illustrate that vapor transition to adjoining wells causes false-positive results of bioassays performed in nonsealed microtiter plates. The microplate layout and a duration of the assay were demonstrated as the key aspects defining level of the results affection by the vapors of volatile agents. Additionally, we reported biological activities and chemical composition of essential oils from A. elegans seeds and X. verdugonianus leaves, which were, according to our best knowledge, analyzed for the first time. Considering our findings, certain modifications of conventional microplate-based assays are necessary (e.g., using EVA Capmat as vapor barrier) to obtain reliable results when biological properties of volatile agents are evaluated.

Maytenus macrocarpa (Ruiz & Pav.) Briq.: Phytochemistry and Pharmacological Activity.

Maytenus macrocarpa (Celastraceae) is a tree native to Amazonia. Its roots, leaves, bark, and combinations of these are used in traditional medicine mainly to treat rheumatism and, to a lesser extent, to heal wounds and to combat bronchitis and diarrhea. To date, mainly triterpenes and dihydro-ß-agarofuran sesquiterpenes were isolated from M. macrocarpa. Extracts and selected pure compounds isolated from the leaves, roots, and stem bark showed antibacterial, antiviral, antiparasitic, anti-inflammatory, and cytotoxic activities in vitro. The aim of this review is to summarize the available ethnobotanical, phytochemical, and pharmacological information about this traditional Amazonian medicinal tree, as well as to attract the attention of phytochemists and pharmacognosists to this potentially interesting source of ethnopharmaceuticals.

Screening of in vitro antimicrobial activity of plants used in traditional Indonesian medicine.

CONTEXT: In many regions of Indonesia, there are numerous traditional herbal preparations for treatment of infectious diseases. However, their antimicrobial potential has been poorly studied by modern laboratory methods. OBJECTIVE: This study investigates in vitro antimicrobial activity of 49 ethanol extracts from 37 plant species used in Indonesian traditional medicine for treatment against Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. MATERIALS AND METHODS: The plants were collected from the Biopharmaca collection garden, Bogor, Indonesia. The plant material was dried, finely grounded, extracted using ethanol, concentrated, and the dried residue was dissolved in 100% DMSO. Antimicrobial activity was determined in terms of a minimum inhibitory concentration (MIC) using a broth microdilution method in 96-well microplates. RESULTS: The extract of Orthosiphon aristatus (Blume) Miq. (Lamiaceae) leaf produced the strongest antimicrobial effect, inhibiting the growth of C. albicans (MIC 128 µg/mL), S. aureus (MIC 256 µg/mL), E. faecalis (MIC 256 µg/mL) and P. aeruginosa (MIC 256 µg/mL). The leaf extract of Woodfordia floribunda Salisb. (Lythraceae) also exhibited significant effect against C. albicans (MIC 128 µg/mL), S. aureus (MIC 256 µg/mL) and E. faecalis (MIC 256 µg/mL). Rotheca serrata (L.) Steane & Mabb. (Lamiaceae) leaf extract inhibited the growth of S. aureus (MIC 256 µg/mL) and C. albicans (MIC 256 µg/mL). DISCUSSION AND CONCLUSIONS: The leaf extract of O. aristatus and W. floribunda exhibited a significant anti-candidal effect. Therefore, both of these plants can serve as prospective source materials for the development of new anti-candidal agents.