Bioassay-guided discovery of antibacterial agents: in vitro screening of Peperomia vulcanica, Peperomia fernandopoioana and Scleria striatinux

Background

The global burden of bacterial infections is high and has been further aggravated by increasing resistance to antibiotics. In the search for novel antibacterials, three medicinal plants: Peperomia vulcanica, Peperomia fernandopoioana (Piperaceae) and Scleria striatinux (Cyperaceae), were investigated for antibacterial activity and toxicity.

Methods

Crude extracts of these plants were tested by the disc diffusion method against six bacterial test organisms followed by bio-assay guided fractionation, isolation and testing of pure compounds. The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were measured by the microdilution method. The acute toxicity of the active extracts and cytotoxicity of the active compound were performed in mice and mammalian cells, respectively.

Results

The diameter of the zones of inhibition (DZI) of the extracts ranged from 7?C13?mm on Escherichia coli and Staphylococcus aureus of which the methylene chloride:methanol [1:1] extract of Scleria striatinux recorded the highest activity (DZI?=?13?mm). Twenty-nine pure compounds were screened and one, Okundoperoxide, isolated from S. striatinux, recorded a DZI ranging from 10?C19?mm on S. aureus. The MICs and MBCs indicated that the Peperomias had broad-spectrum bacteriostatic activity. Toxicity tests showed that Okundoperoxide may have a low risk of toxicity with an LC₅₀ of 46.88???g/mL.

Conclusions

The antibacterial activity of these plants supports their use in traditional medicine. The pure compound, Okundoperoxide, may yield new antibacterial lead compounds following medicinal chemistry exploration.     

Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection

Background

Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated.

Results

Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material.

Conclusion

Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.     

Antibiotic-resistant bacteria in the guts of insects feeding on plants: prospects for discovering plant-derived antibiotics

Background

Although plants produce many secondary metabolites, currently none of these are commercial antibiotics. Insects feeding on specific plants can harbour bacterial strains resistant to known antibiotics suggesting that compounds in the plant have stimulated resistance development. We sought to determine whether the occurrence of antibiotic-resistant bacteria in insect guts was a widespread phenomenon, and whether this could be used as a part of a strategy to identify antibacterial compounds from plants.

Results

Six insect/plant pairs were selected and the insect gut bacteria were identified and assessed for antibiotic susceptibilities compared with type strains from culture collections. We found that the gut strains could be more or less susceptible to antibiotics than the type strains, or show no differences. Evidence of antibacterial activity was found in the plant extracts from five of the six plants, and, in one case Catharanthus roseus (Madagascar Periwinkle), compounds with antibacterial activity were identified.

Conclusion

Bacterial strains isolated from insect guts show a range of susceptibilities to antibiotics suggesting a complex interplay between species in the insect gut microbiome. Extracts from selected plants can show antibacterial activity but it is not easy to isolate and identify the active components. We found that vindoline, present in Madagascar Periwinkle extracts, possessed moderate antibacterial activity. We suggest that plant-derived antibiotics are a realistic possibility given the advances in genomic and metabolomic methodologies.

     

Thiosulphate-induced mercury accumulation by plants: metal uptake and transformation of mercury fractionation in soil - results from a field study

Aims

The thiosulphate induced accumulation of mercury by the three plants Brassica juncea var.LDZY, Brassica juncea var.ASKYC and Brassica napus var. ZYYC and the transformation of mercury fractionation in the rhizosphere of each plant was investigated in the field.

Methods

Experimental farmland was divided into control and thiosulphate plots. Each plot was divided into three subplots with each planted with one of the plants. After harvesting, the mercury concentration in plants, mercury fractionation in rhizosphere soil before and after phytoextraction, and the vertical distribution of bioavailable mercury in bulk soil profiles was analyzed.

Results

The cultivar B. juncea var.LDZY accumulated a higher amount of mercury in shoots than the other two plants. Thiosulphate treatment promoted an increase in the concentration of metal in plants and a transformation of Fe/Mn oxide-bound and organic-bound mercury (potential bioavailable fractions) into soluble and exchangeable and specifically-sorbed fractions in the rhizosphere. The observed increase in bioavailable rhizosphere mercury concentration was restricted to the root zone; mercury did not move down the soil profile as a function of thiosulphate application to soil.

Conclusions

Thiosulphate-induced phytoextraction has the potential to manage environmental risk of mercury in soil by decreasing the concentration of mercury associated with potential bioavailable fraction that can be accumulated by crop plants.     

Variation of secondary metabolite levels in maize seedling roots induced by inoculation with Azospirillum, Pseudomonas and Glomus consortium under field conditions

Background and aims

Many plant-beneficial microorganisms can influence secondary plant metabolism, but whether these effects add up when plants are co-inoculated is unclear. This issue was assessed, under field conditions, by comparing the early impacts of seed inoculation on secondary metabolite profiles of maize at current or reduced mineral fertilization levels.

Methods

Maize seeds were inoculated singly with selected strains from bacterial genera Pseudomonas and Azospirillum or mycorrhizal genus Glomus, or with these strains combined two by two or all three together. At 16?days, maize root methanolic extracts were analyzed by RP-HPLC and secondary metabolites (phenolics, flavonoids, xanthones, benzoxazionoids, etc.) identified by LC/MS.

Results

Inoculation did not impact on plant biomass but resulted in enhanced total root surface, total root volume and/or root number in certain inoculated treatments, at reduced fertilization. Inoculation led to qualitative and quantitative modifications of root secondary metabolites, particularly benzoxazinoids and diethylphthalate. These modifications depended on fertilization level and microorganism(s) inoculated. The three selected strains gave distinct results when used alone, but unexpectedly all microbial consortia gave somewhat similar results.

Conclusions

The early effects on maize secondary metabolism were not additive, as combining strains gave effects similar to those of Glomus alone. This is the first study demonstrating and analyzing inoculation effects on crop secondary metabolites in the field.     

Characterization of the antiproliferative activity of Xylopia aethiopica

Background

Xylopia aethiopica, a plant found throughout West Africa, has both nutritional and medicinal uses. The present study aims to characterize the effects of extracts of this plant on cancer cells.

Results

We report that X. aethiopica extract prepared with 70% ethanol has antiproliferative activity against a panel of cancer cell lines. The IC50 was estimated at 12 ??g/ml against HCT116 colon cancer cells, 7.5 ??g/ml and > 25 ??g/ml against U937 and KG1a leukemia cells, respectively. Upon fractionation of the extract by HPLC, the active fraction induced DNA damage, cell cycle arrest in G1 phase and apoptotic cell death. By using NMR and mass spectrometry, we determined the structure of the active natural product in the HPLC fraction as ent-15-oxokaur-16-en-19-oic acid.

Conclusion

The main cytotoxic and DNA-damaging compound in ethanolic extracts of Xylopia aethiopica is ent-15-oxokaur-16-en-19-oic acid.     

Possible contribution of Bradyrhizobium on nitrogen fixation in sweet potatoes

Aims

Sweet potato (Ipomoea batatas) is known for its ability to grow under nitrogen-limited conditions. To clarify the possible contribution of biological nitrogen fixation, we tried to isolate and identify diazotrophic bacteria from sweet potatoes.

Methods

By using cultivation technique, we isolated putative endophytes, which possess nifH genes, from surface-sterilized sweet potatoes. Their nitrogen-fixing abilities were demonstrated by the acetylene reduction assay in a semi-solid malate medium and sweet potato extracts. We also examined the colonization of an isolated strain (AT1) in sweet potatoes and their influence on growth and nitrogen fixation in plants as assessed by an acetylene reduction assay and ¹⁵N-isotope dilution technique.

Results

The isolates were identified as strains of Bradyrhizobium sp. AT1, Paenibacillus sp. AS2 and Pseudomonas sp. T16 based on their 16S rRNA gene sequences. They showed acetylene reduction activity (ARA) in the semi-solid malate medium. Among them, B. sp. AT1 showed ARA in sweet potato extracts under micro-aerobic conditions whereas both P. sp. AS2 and P. sp. T16 showed no ARA. The inoculation of B. sp. AT1 to the sweet potatoes resulted in increases in the fresh weights and detection of ARA in the inoculated plants. Moreover, the reduction of ¹⁵N atom % was observed in the inoculated plants compared to uninoculated controls.

Conclusions

B. sp. AT1 actively expresses nitrogenase activity in sweet potatoes and may contribute to the nitrogen nutrition of host plants.     

Earlywood vessels of the sub-Mediterranean oak Quercus pyrenaica have greater plasticity and sensitivity than those of the temperate Q. petraea at the Atlantic–Mediterranean boundary

Key message

Earlywood vessel features indicate different adaptations of Quercus petraea and Q. pyrenaica , which are probably related with their corresponding Atlantic and sub-Mediterranean ecological requirements.

Abstract

We studied the climatic signal of the earlywood anatomy of a temperate [Quercus petraea (Mattuschka) Liebl.] and a sub-Mediterranean (Quercus pyrenaica Willd.) oak species growing under similar climatic conditions in a transitional area between the Atlantic and Mediterranean regions of the Iberian Peninsula. We hypothesized that both species react differently in their wood anatomy due to their contrasting ecological requirements, and we test the usefulness of earlywood anatomical features to study the behaviour of these ring-porous oaks upon climate. For this, we measured the earlywood vessels, and obtained annual series of several anatomical variables for the period 1937–2006 using dendrochronological techniques, considering whether the vessels belonged to the first row or not. After optimizing the data set by principal component analysis and progressive filtering of large vessels, we selected maximum vessel area and total number of vessels as they resulted to be the optimal variables to describe vessel size and number, respectively. Vessel size of Q. pyrenaica was dependent on precipitation along the previous growing season, whereas it did not show any clear climatic response for Q. petraea. On the contrary, vessel number was related to winter temperature for both species. These relationships observed between climate and anatomy appeared to be stable through time. The results obtained reinforce the utility of earlywood vessel features as potential climate proxies.     

Spatial root distribution of plants growing in vertical media for use in living walls

Background and Aims

For plants growing in living walls, the growth potential is correlated to the roots ability to utilize resources in all parts of the growing medium and thereby to the spatial root distribution. The aim of the study was to test how spatial root distribution was affected by growing medium, planting position and competition from other plants.

Methods

Five species (Campanula poscharskyana cv. ‘Stella’, Fragaria vesca cv. ‘Småland’, Geranium sanguineum cv. ‘Max Frei’, Sesleria heufleriana and Veronica officinalis cv. ‘Allgrün’) were grown in three growing media (coir and two of rockwool) in transparent boxes under greenhouse conditions. Root frequency was registered and the activity of individual root systems was studied via ¹⁵N uptake and plant dry weight was measured.

Results

Plants in coir had stronger root growth in all parts of the medium than plants in rockwool. Upwards root growth was limited for plants in the middle or lower parts of the medium and ¹⁵N measurements confirmed that only plants in the bottom of the box had active roots in the bottom of the medium. The species differed in root architecture and spatial root distribution.

Conclusions

The choice of growing medium, plant species and planting position is important for a living wall as it affects the spatial root growth of the plants.     

Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.     

Effects of antibacterial mineral leachates on the cellular ultrastructure, morphology, and membrane integrity of Escherichia coli and methicillin-resistant Staphylococcus aureus

Background

We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates.

Methods

We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Bac light LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity.

Results

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates.

Conclusions

These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells.     

Use of a mixed culture strategy to isolate halophilic bacteria with antibacterial and cytotoxic activity from the Manaure solar saltern in Colombia

Background

Water evaporation in solar salterns creates salinity gradients that promote the adaptation of microbial species to different salinities. This competitive habitat challenges the metabolic capabilities of microorganisms and promotes alterations in their production of secondary metabolites. Thus, solar salterns are a potentially important source of new natural products. In Colombia, the most important and representative solar saltern is located in Manaure (La Guajira) in the north of Colombia. The aim of this study was to develop an alternative screening strategy to select halophilic bacteria as producers of bioactive compounds from mixed microbial cultures rather than individual environmental isolates. Brine and sediment samples from different ponds (across a salinity gradient) were inoculated in seven different culture media to grow bacteria and archaea, allowing for a total of 40 different mixed cultures. An organic extract from each mixed culture was obtained and tested against multidrug resistant pathogens, including Klebsiella pneumoniae, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus and Bacillus subtilis. In addition, the extracts were tested against two human cancer cell lines, cervical adenocarcinoma (SiHa) and lung carcinoma (A-549).

Results

Twenty-four of the forty extracts from mixed cultures obtained from brine and sediment samples from the Manaure solar saltern showed antibacterial activity against Bacillus subtilis. Two extracts, referred to as A1SM3–29 and A1SM3–36, were also active against a methicillin-resistant Staphylococcus aureus, with the latter extract also showing slight cytotoxic activity against the assayed human lung cancer cell line. From this mixed culture, nine isolates were cultivated, and their extracts were tested against the same pathogens, resulting in the identification of a Vibrio sp. strain (A1SM3–36-8) with antimicrobial activity that was similar to that observed for the mixed culture extract. The extract of this strain was subjected to a bioautography assay, and 3 different fractions exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. Based on the amount obtained for each fraction, F3 was selected to isolate and identify its metabolites. The major compound was identified by NMR and HRMS as 13-cis-docosenamide, an amide that has been previously reported to be an antimicrobial and cytotoxic compound.

Conclusions

Our results shows the utility of our strategy in detecting bioactive molecules in initial mixed cultures by biological assays, resulting in the isolation and characterization of Vibrio sp. A1SM3–36-8, a halophilic strain with great antibacterial and cytotoxic potential.

     

Competitive interaction between the exotic plant Rhus typhina L. and the native tree Quercus acutissima Carr. in Northern China under different soil N:P ratios

Background and aims

Anthropogenic nitrogen (N) and phosphorus (P) input has changed the relative importance of nutrient elements. This study aimed to examine the effects of different nutrient conditions on the interaction between exotic and native plants.

Methods

We conducted a greenhouse experiment with a native species Quercus acutissima Carr. and an exotic species Rhus typhina L. grown in monocultures or mixtures, under three N:P ratios (5, 15 and 45 corresponding to N-limited, basic N and P supply and P-limited conditions, respectively). After 12 weeks of treatment, traits related to biomass allocation, leaf physiology and nutrient absorption were determined.

Results

R. typhina was dominant under competition, with a high capacity for carbon assimilation and nutrient absorption, and the dominance was unaffected by increasing N:P ratios. R. typhina invested more photosynthate in leaves and more nutrients in the photosynthetic apparatus, enabling high biomass production. Q. acutissima invested more photosynthate in roots and more nutrients in leaf persistence at the expense of reduced carbon assimilation capacity.

Conclusions

Different trade-offs in biomass and nutrient allocation of the two species is an important reason for their distinct performances under competition and helps R. typhina to maintain dominance under different nutrient conditions.     

Mutation of mpk6 enhances cadmium tolerance in Arabidopsis plants by alleviating oxidative stress

Background and aims

Cadmium (Cd) could activate activity of mitogen-activated protein kinase MPK6 in plants. In this study, we investigated the role of MPK6 in mediating Cd toxicity in plants.

Methods

The wild type Arabidopsis plants (WT) and the mpk6-2 mutants were subjected either 0 (Control) or 10 μM Cd treatment. Kinase activity of MPK6, nitric oxide (NO) level, Cd concentration, and oxidative stress were measured.

Results

In WT plants, Cd exposure rapidly stimulated kinase activity of MPK6. However, upon Cd exposure, mpk6-2 showed better growth than the WT. Although Cd-induced production of NO in roots was greater in WT than in mpk6-2, there was no difference in Cd concentration between the two plants. Nevertheless, the Cd-induced hydroperoxide burst, lipid peroxidation and loss of membrane integrity, were all more severe in the WT than in mpk6-2. Foliar applications of antioxidant ascorbic acid, vigorously improved the growth of both the WT and mpk6-2 under Cd exposure. Thereby the growth difference between these two plants was minimized.

Conclusions

Mutation of mpk6 enhances Cd tolerance in plants by alleviating oxidative stress, but did not affect cadmium accumulation in plants.     

Plant chitinase responses to different metal-type stresses reveal specificity

Key message

Chitinases in Glycine max roots specifically respond to different metal types and reveal a polymorphism that coincides with sensitivity to metal toxicity.

Abstract

Plants evolved various defense mechanisms to cope with metal toxicity. Chitinases (EC 3.2.1.14), belonging to so-called pathogenesis-related proteins, act as possible second line defense compounds in plants exposed to metals. In this work their activity was studied and compared in two selected soybean (Glycine max L.) cultivars, the metal-tolerant cv. Chernyatka and the sensitive cv. Kyivska 98. Roots were exposed to different metal(loid)s such as cadmium, arsenic and aluminum that are expected to cause toxicity in different ways. For comparison, a non-metal, NaCl, was applied as well. The results showed that the sensitivity of roots to different stressors coincides with the responsiveness of chitinases in total protein extracts. Moreover, detailed analyses of acidic and neutral proteins identified one polymorphic chitinase isoform that distinguishes between the two cultivars studied. This isoform was stress responsive and thus could reflect the evolutionary adaptation of soybean to environmental cues. Activities of the individual chitinases were dependent on metal type as well as the cultivar pointing to their more complex role in plant defense during this type of stress.     

Dereplication of natural products from complex extracts by regression analysis and molecular networking: case study of redox-active compounds from <Emphasis Type="Italic">Viola alba</Emphasis> subsp. <Emphasis Type="Italic">dehnhardtii</Emphasis>

Introduction

In natural product research, bioassay-guided fractionation was previously widely employed but is now judged to be inadequate in terms of time and cost, particularly if only known compounds are ultimately isolated. The development of metabolomics, along with improvements in analytical tools, allows comprehensive metabolite profiling. This enables dereplication to target unknown active compounds early in the purification workflow.

Objectives

Starting from an ethanolic extract of violet leaves, this study aims to predict redox active compounds within a complex matrix through an untargeted metabolomics approach and correlation analysis.

Methods

Rapid fractionation of crude extracts was carried out followed by multivariate data analysis (MVA) of liquid chromatography–high resolution mass spectrometry (LC–HRMS) profiles. In parallel, redox active properties were evaluated by the capacity of the molecules to reduce 2,2-diphenyl-1-picrylhydrazyl (DPPH^·) and superoxide (O₂ ^·?) radicals using UV–Vis and electron spin resonance spectroscopies (ESR), respectively. A spectral similarity network (molecular networking) was used to highlight clusters involved in the observed redox activities.

Results

Dereplication on Viola alba subsp. dehnhardtii highlighted a reproducible pool of redox active molecules. Polyphenols, particularly O-glycosylated coumarins and C-glycosylated flavonoids, were identified and de novo dereplicated through molecular networking. Confirmatory analyses were undertaken by thin layer chromatography (TLC)–DPPH–MS assays and nuclear magnetic resonance (NMR) spectra of the most active compounds.

Conclusion

Our dereplication strategy allowed the screening of leaf extracts to highlight new biologically active metabolites in few steps with a limited amount of crude material and reduced time-consuming manipulations. This approach could be applied to any kind of natural extract for the study of various biological activities.

     

Characterization of the sugar-O-methyltransferase LobS1 in lobophorin biosynthesis

Lobophorins A (1) and B (2) belong to a large group of spirotetronate natural products with potent antibacterial and antitumor activities. The cloning of the lobophorin biosynthesis gene cluster from the deep-sea-derived Streptomyces sp. SCSIO 01127 identified a sugar-O-methyltransferase-encoding gene lobS1. The lobS1 inactivation mutant accumulated two new lobophorin analogs 3 and 4, different from 1 and 2 by lacking the 4-methyl group at the terminal l-digitoxose, respectively. Biochemical experiments verified that LobS1 was a SAM-dependent sugar-O-methyltransferase that required divalent metal ions for better activity. Antibacterial assays revealed compounds 3 and 4 were generally less potent than compounds 1 and 2. These findings suggest that the methylation on the terminal digitoxose by LobS1 tailors lobophorin biosynthesis and highlights the importance of this methylation for antibacterial potence.