Salvia officinalis L., Sambucus nigra L., Matricaria chamomilla L., Agrimonia eupatoria L., Fragaria vesca L. and Malva sylvestris L. are plants that have a long tradition in European folk medicine. To this day, they are part of medicinal teas or creams that help with the healing of skin wounds and the treatment of respiratory or intestinal infections. However, so far these plants have not been investigated more deeply than in their direct antibacterial effect. Our research is focused on adjuvants that inhibit the mechanism of antibiotic resistance or modulate bacterial virulence. Based on a preliminary screening of 52 European herbs, which commonly appear as part of tea blends or poultice. Six of them were selected for their ability to revert the resistant phenotype of nosocomial bacterial strains. Herbs selected for this study were obtained from commercially available sources. For the extraction of active compounds ethanol was used. Modulation of virulence was observed as an ability to inhibit bacterial cell-to-cell communication using two mutant sensor strains of Vibrio campbellii. Biofilm formation, and planktonic cell adhesion was measured using a static antibiofilm test. Ethidium bromide assay was used to checked the potential of inhibition bacterial efflux pumps. The antibacterial activities of the herbs were evaluated against resistant bacterial strains using macro dilution methods. Alcohol extracts had antibacterial properties mainly against Gram-positive bacteria. Of all of them, the highest antimicrobial activity demonstrated Malva sylvestris, killing both antibiotic resistant bacteria; Staphylococcus aureus with MIC of 0.8 g/L and Pseudomonas aeruginosa 0.7 g/L, respectively. Fragaria vesca extract (0.08 g/L) demonstrated strong synergism with colistin (4 mg/L) in modulating the resistant phenotype to colistin of Pseudomonas aeruginosa. Similarly, the extract of S. officinalis (0.21 g/L) reverted resistance to gentamicin (1 mg/L) in S. aureus. However, Sambucus nigra and Matricaria chamomilla seem to be a very promising source of bacterial efflux pump inhibitors. The extract of F. vesca was the most active. It was able to reduce biofilm formation probably due to the ability to decrease bacterial quorum sensing. On the other hand, the activity of S. nigra or M. chamomilla in reducing bacterial virulence may be explained by the ability to inhibit bacterial efflux systems. All these plants have potential as an adjuvant for the antibiotic treatment.

Antibiotic resistance is currently a serious health problem. Since the discovery of new antibiotics no longer seems to be a sufficient tool in the fight against multidrug-resistant infections, adjuvant (combination) therapy is gaining in importance as well as reducing bacterial virulence. Silymarin is a complex of flavonoids and flavonolignans known for its broad spectrum of biological activities, including its ability to modulate drug resistance in cancer. This work aimed to test eleven, optically pure silymarin flavonolignans for their ability to reverse the multidrug resistance phenotype of Staphylococcus aureus and reduce its virulence. Silybin A, 2,3-dehydrosilybin B, and 2,3-dehydrosilybin AB completely reversed antibiotic resistance at concentrations of 20 µM or less. Both 2,3-dehydrosilybin B and AB decreased the antibiotic-induced gene expression of representative efflux pumps belonging to the major facilitator (MFS), multidrug and toxic compound extrusion (MATE), and ATP-binding cassette (ABC) families. 2,3-Dehydrosilybin B also inhibited ethidium bromide accumulation and efflux in a clinical isolate whose NorA and MdeA overproduction was induced by antibiotics. Most of the tested flavonolignans reduced cell-to-cell communication on a tetrahydrofuran-borate (autoinducer-2) basis, with isosilychristin leading the way followed by 2,3-dehydrosilybin A and AB, which halved communication at 10 µM. Anhydrosilychristin was the only compound that reduced communication based on acyl-homoserine lactone (autoinducer 1), with an IC₅₀ of 4.8 µM. Except for isosilychristin and anhydrosilychristin, all of the flavonolignans inhibited S. aureus surface colonization, with 2,3-dehydrosilybin A being the most active (IC₅₀ 10.6 µM). In conclusion, the selected flavonolignans, particularly derivatives of 2,3-dehydrosilybin B, 2,3-dehydrosilybin AB, and silybin A are non-toxic modulators of S. aureus multidrug resistance and can decrease the virulence of the bacterium, which deserves further detailed research.

Selaginella P. Beauv. is a group of vascular plants in the family Selaginellaceae Willk., found worldwide and numbering more than 700 species, with some used as foods and medicines. The aim of this paper was to compare methanolic (MeOH) and dichloromethane (DCM) extracts of eight Selaginella species on the basis of their composition and biological activities. Six of these Selaginella species are underinvestigated. Using ultra-high performance liquid chromatography–high-resolution mass spectrometry (UHPLC–HRMS) analysis, we identified a total of 193 compounds among the tested Selaginella species, with flavonoids predominating. MeOH extracts recovered more constituents that were detected, including selaginellins, the occurrence of which is only typical for this plant genus. Of all the tested species, Selaginellaapoda contained the highest number of identified selaginellins. The majority of the compounds were identified in S. apoda, the fewest compounds in Selaginellacupressina. All the tested species demonstrated antioxidant activity using oxygen radical absorption capacity (ORAC) assay, which showed that MeOH extracts had higher antioxidant capacity, with the half maximal effective concentration (EC₅₀) ranging from 12 ± 1 (Selaginellamyosuroides) to 124 ± 2 (Selaginellacupressina) mg/L. The antioxidant capacity was presumed to be correlated with the content of flavonoids, (neo)lignans, and selaginellins. Inhibition of acetylcholinesterase (AChE) was mostly discerned in DCM extracts and was only exhibited in S. myosuroides, S. cupressina, Selaginellabiformis, and S. apoda extracts with the half maximal inhibitory concentration (IC₅₀) in the range of 19 ± 3 to 62 ± 1 mg/L. Substantial cytotoxicity against cancer cell lines was demonstrated by the MeOH extract of S. apoda, where the ratio of the IC₅₀ HEK (human embryonic kidney) to IC₅₀ HepG2 (hepatocellular carcinoma) was 7.9 ± 0.2. MeOH extracts inhibited the production of nitrate oxide and cytokines in a dose-dependent manner. Notably, S. biformis halved the production of NO, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 at the following concentrations: 105 ± 9, 11 ± 1, and 10 ± 1 mg/L, respectively. Our data confirmed that extracts from Selaginella species exhibited cytotoxicity against cancer cell lines and AChE inhibition. The activity observed in S. apoda was the most promising and is worth further exploration.

(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.

Overexpression of the NorA efflux pump is a principal mechanism by which Staphylococcus aureus pumps out fluoroquinolone antibiotics. Therefore, making efflux pump inhibitors (EPIs) is a compelling strategy to restore antibiotic efficacy. Sulphur containing heterocycles and peptidomimetic scaffolds such as Phe-Arg-β-naphthylamide (PAβN) have independently demonstrated NorA EPI activity, yet their clinical translation has been hampered by cytotoxicity at active concentrations. To merge these two chemotypes, herein we report the design and synthesis of three peptidomimetic libraries containing thiophene-3-carboxylic acyl (I), 3-(methylthio)propanoic acyl (II) and 3-(methylsulfinyl)propanoic acyl (III) moieties to investigate the contribution of ring rigidity, aromaticity and sulphur oxidation state to EPI potency. Using fluorescence-based ethidium bromide (EtBr) accumulation assay, broth microdilution and checkerboard assays, we assessed 42 sulphur peptidomimetics for their ability to inhibit NorA efflux pump in S. aureus. Lead compounds 3f and 3g demonstrated potent EPI activity in a NorA overexpressing S. aureus strain and checkerboard assays showed synergistic/additive effects in combination with ciprofloxacin and norfloxacin resulting in meaningful reductions of minimal inhibitory concentration (MIC). Furthermore, molecular docking was used to determine the most favourable interactions and binding pose. Systematic structure-activity relationship (SAR) analysis and molecular docking study revealed that the rigid thiophene ring and 4-methoxy benzyl isocyanide component are both critical determinants of the inhibitory activity. Replacement of these fragments or oxidized sulfinyl analogues led to significant loss of potency. Crucially, both compounds exhibited no cytotoxicity toward healthy human cells up to 200 μM, overcoming the key limitation of existing EPIs. In general, these results uphold the idea that rationally designed peptidomimetic frameworks can act as competitive inhibitors of bacterial efflux pumps and may work as potential antibiotic adjuvants to reduce efflux-driven resistance.