Cyclotides are cyclic plant polypeptides of 27–37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less is known about the relevance of cyclotides for the plants producing them. The anti-insect larvae effects of kB1 and antibacterial activity of cyO2 suggest that cyclotides are a part of plant host defense. The sweet violet (Viola odorata L.) produces a wide array of cyclotides, including kB1 (kalata B1) and cyO2 (cycloviolacin O2), with distinct presumed biological roles. Here, we evaluate V. odorata cyclotides’ potency against plant pathogens and their mode of action using bioassays, liposome experiments and immunogold labeling for transmission electron microscopy (TEM). We explore the link between the biological activity and distribution in plant generative, vegetative tissues and seeds, depicted by immunohistochemistry and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Cyclotides cyO2, cyO3, cyO13, and cyO19 are shown to have potent activity against model fungal plant pathogens (Fusarium oxysporum, F. graminearum, F. culmorum, Mycosphaerella fragariae, Botrytis cinerea) and fungi isolated from violets (Colletotrichum utrechtense and Alternaria alternata), with minimal inhibitory concentrations (MICs) ranging from 0.8 μM to 25 μM. Inhibition of phytopathogenic bacteria – Pseudomonas syringae pv. syringae, Dickeya dadantii and Pectobacterium atrosepticum – is also observed with MIC = 25–100 μM. A membrane-disrupting antifungal mode of action is shown. Finding cyO2 inside the fungal spore cells in TEM images may indicate that other, intracellular targets may be involved in the mechanism of toxicity. Fungi can not break down cyclotides in the course of days. varv A (kalata S) and kB1 show little potency against pathogenic fungi when compared with the tested cycloviolacins. cyO2, cyO3, cyO19 and kB1 are differentially distributed and found in tissues vulnerable to pathogen (epidermis, rizodermis, vascular bundles, protodermis, procambium, ovary walls, outer integuments) and pest (ground tissues of leaf and petiole) attacks, respectively, indicating a link between the cyclotides’ sites of accumulation and biological role. Cyclotides emerge as a comprehensive defense system in V. odorata, in which different types of peptides have specific targets that determine their distribution in plant tissues.

Staphylococcus aureus is the most common infectious agent involved in the development of skin infections that are associated with antibiotic resistance, such as burn wounds. As drug resistance is a growing problem it is essential to establish novel antimicrobials. Currently, antibiotic resistance in bacteria is successfully controlled by multi-drug therapies. Here we demonstrate that secondary metabolites present in the extract obtained from Drosera binata in vitro cultures are effective antibacterial agents against S. aureus grown in planktonic culture and in biofilm. Moreover, this is the first report demonstrating the synergistic interaction between the D. binata extract and silver nanoparticles (AgNPs), which results in the spectacular enhancement of the observed bactericidal activity, while having no cytotoxic effects on human keratinocytes. Simultaneous use of these two agents in significantly reduced quantities produces the same effect, i.e. by killing 99.9% of bacteria in inoculum or eradicating the staphylococcal biofilm, as higher amounts of the agents used individually. Our data indicates that combining AgNPs with either the D. binata extract or with its pure compound (3-chloroplumbagin) may provide a safe and highly effective alternative to commonly used antibiotics, which are ineffective towards the antibiotic-resistant S. aureus.

Pseudomonas aeruginosa is a common causative bacterium of acute and chronic infections that have been responsible for high mortality over the past decade. P. aeruginosa produces many virulence factors such as toxins, enzymes and dyes that are strongly dependent on quorum sensing (QS) signaling systems. P. aeruginosa has three major QS systems (las, rhl and Pseudomonas quinolone signal) that regulate the expression of genes encoding virulence factors as well as biofilm production and maturation. Antimicrobial blue light (aBL) is considered a therapeutic option for bacterial infections and has other benefits, such as reducing bacterial virulence. Therefore, this study investigated the efficacy of aBL to reduce P. aeruginosa pathogenicity. aBL treatment resulted in the reduced activity of certain QS signaling molecules in P. aeruginosa and inhibited biofilm formation. in vivo tests using a Caenorhabditis elegans infection model indicated that sublethal aBL decreased the pathogenicity of P. aeruginosa. aBL may be a new virulence‐targeting therapeutic approach.

Possibilities to protect potato tubers from rotting caused by Soft Rot Pectobacteriaceae (SRP) under disease favoring conditions were investigated using compatible mixtures of bacterial antagonists and tested with a newly developed stepwise efficacy-based screening protocol. Twenty-two bacterial antagonists were evaluated against a combination of five Pectobacterium and Dickeya strains representing species and subspecies most often associated with potato soft rot in Europe. To enable potential synergistic activity, the antagonists were initially tested against the combination of pathogens in 15 random mixtures containing up to 5 antagonists each. Three mixtures (M2, M4, and M14) out of 15 tested reduced tuber tissue maceration due to soft rot. The individual antagonists derived from M2, M4, and M14 mixtures were tested on potato slices and whole tuber injection assays. These five strains (S. plymuthica strain A294, E. amnigenus strain A167, R. aquatilis strain H145, S. rubidaea strain H440, and S. rubidaea strain H469) were combined to develop a tailored biological control mixture against potato soft rot. The new mixture, designated the Great Five (GF), was tested on seed potato tubers vacuum infiltrated with antagonists and subsequently with the combination of five SRP pathogens. In these experiments, the GF mixture provided stable protection of inoculated potato tubers, reducing soft rot by 46% (P = 0.0016) under high disease pressure conditions. The A294, A167, H145, H440, and H469 antagonists were characterized for features important for viable commercial applications including growth at different temperatures, resistance to antibiotics, and potential toxicity toward Caenorhabditis elegans. The implications for control of soft rot caused by SRP with the use of the GF mixture of antagonists are discussed.

With the rapid and continuous emergence of antimicrobial resistance, bacterial infections became a significant global healthcare concern. One of the proposed strategies to combat multidrug-resistant pathogens is to use additional compounds, such as natural biologically active substances, as adjuvants for existing antibiotics. In this study, we investigated the potential of caffeine, the widely consumed alkaloid, to modulate the antibacterial effects of antibiotics commonly used in clinical practice. We used disc diffusion assay to evaluate the effects of caffeine on 40 antibiotics in two Staphylococcus aureus strains (methicillin-resistant and methicillin-sensitive). Based on the results of this step, we selected five antibiotics for which the greatest caffeine-induced improvements in antibacterial activity were observed, and further analyzed their interactions with caffeine using a checkerboard approach. Caffeine at concentrations of 250 µg/mL or higher halved the MIC values of ticarcillin, cefepime, gentamycin, azithromycin, and novobiocin for all gram-negative species investigated (Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii). At the highest caffeine concentrations tested (up to 16 mg/mL), decreases in MIC values were 8- to 16-fold. The obtained results prove that caffeine modulates the activity of structurally diverse antibiotics, with the most promising synergistic effects observed for cefepime and azithromycin toward gram-negative pathogens.