Control of the Verticillium Wilt on Tomato Plants by Means of Olive Leaf Extracts Loaded on Chitosan Nanoparticles

In this research, a new ecofriendly and sustainable fungicide agent, with the ability to control Verticillium wilt, was developed. To this purpose, a green extract of olive leaf (OLE) was prepared by ultrasound-assisted extraction (UAE) and characterized in terms of polyphenol content and antioxidant activity. Then, OLE was loaded in chitosan nanoparticles (CTNPs) to combine the antifungal activity of CTNPs and phenolic compounds to obtain an important synergic effect. Nanoparticles were synthetized using the ionic gelation technique and characterized in terms of sizes, polydispersity index, Z-potential, encapsulation efficiency, and release profile. Qualitative and quantitative analyses of OLE were performed by the HPLC method. OLE-loaded CTNPs exhibited good physicochemical properties, such as a small size and positive surface charge that significantly contributed to a high antifungal efficacy against Verticillum dahliae. Therefore, their antifungal activity was evaluated in vitro, using the minimal inhibition concentration (MIC) assay in a concentration range between 0.071 and 1.41 mg/mL. Free OLE, blank CTNPs, and OLE-loaded CTNPs possessed MIC values of 0.35, 0.71, and 0.14 mg/mL, respectively. These results suggest an important synergic effect when OLE was loaded in CTNPs. Thereafter, we tested the two higher concentrations on tomato plants inoculated with V. dahliae, where no fungal growth was observed in the in vitro experiment, 0.71 and 1.41 mg/mL. Interestingly, OLE-loaded CTNPs at the higher concentration used, diminished the symptoms of Verticillium wilt in tomato plants inoculated with V. dahliae and significantly enhanced plant growth. This research offers promising results and opens the possibility to use OLE-loaded CTNPs as safe fungicides in the control strategies of Verticillium wilt at open field.

Catalytic and Biological Activity of Silver and Gold Complexes Stabilized by NHC with Hydroxy Derivatives on Nitrogen Atoms

In this paper is reported the synthesis of N,N′ hydroxy derivative of NHC silver (3a–4a) and gold(I) (3b–4b) complexes of general formula [M(NHC)2]+ [MX2]−. All compounds were characterized by spectroscopic and analytic techniques. The complexes turned out to be effective in both catalytic and biological applications. They catalyzed the coupling of aldehyde, piperidine, and phenylacetylene in A3-reaction to produce propargylamines and showed antimicrobial activity. In fact, minimal inhibition concentration (MIC) tests with Gram-positive and Gram-negative bacteria demonstrated that the silver compounds are selective toward E. coli, whereas the gold analogues are active against S. aureus. Moreover, the N,N′ hydroxy derivative of NHC silver complexes 3a and 4a exhibited good anticancer activity on the HeLA cancer cells (3a-IC50 = 12.2 ± 0.1 µM, 4a-IC50 = 11.9 ± 1.2 µM), whereas gold complex 4b displayed good anticancer activity towards the MCF-7 cells (IC50 = 12.2 ± 1.2 µM).

Study on the In vitro and In vivo Antifungal Activities of Nano-silver against Mycoleptodiscus indicus causing Leaf Blight on Lotus in Vietnam

Background: Lotus, Nelumbo nucifera Gaertn is Vietnam’s symbol and Buddhism’s flower and plays an essential part in rural Vietnam’s economy as all aspects of lotus could bring benefits to farmers. But, unfortunately, lotus yield in Vietnam is seriously affected by various plant diseases. Among them, leaf blight is currently emerging as one of the primary diseases devastating lotus crops in Vietnam, in which there are large necrotic parts on lotus leaves, flowers and seeds. Methods: Aloe barbadensis extract was used for synthesizing silver nanoparticles. Leaf blight lotus leaves were collected to isolate pathogenic fungi-infection of isolated pathogenic fungi on the healthy lotus and then identify hidden mold by 28S rRNA sequencing. Determination of in vitro minimal inhibition concentration of nano-silver was conducted according to Azizi. The reduction of disease symptoms and biological characteristics of the treated lotus was observed. Result: Morphological analysis and molecular identification of 28S rRNA sequencing showed that the pathogenic microorganism was Mycoleptodiscus indicus (M. indicus). Both in vitro antifungal activity and in vivo treatment of leaf blight lotus using a nano-silver solution showed that 30 ppm of nano-silver was the minimal inhibition concentration (MIC) for totally eradicating M. indicus growth. This was the first time M. indicus was reported to infect and cause leaf blight on a lotus. Previously, M. indicus was a well-known plant pathogen that could cross-kingdom infect humans and animals. Thus, the fact that lotus is widely cultured in Vietnamese rural could increase the chance for M. indicus to spread; hence, this raised the alarm about its potential harm to plants, humans and animals. And, significantly, it revealed nano-silver as a possible approach to prevent M. indicus.

Hedgehog artificial macrophage with atomic-catalytic centers to combat Drug-resistant bacteria

Pathogenic drug-resistant bacteria represent a threat to human health, for instance, the methicillin-resistant Staphylococcus aureus (MRSA). There is an ever-growing need to develop non-antibiotic strategies to fight bacteria without triggering drug resistance. Here, we design a hedgehog artificial macrophage with atomic-catalytic centers to combat MRSA by mimicking the “capture and killing” process of macrophages. The experimental studies and theoretical calculations reveal that the synthesized materials can efficiently capture and kill MRSA by the hedgehog topography and substantial generation of •O2− and HClO with its Fe2N6O catalytic centers. The synthesized artificial macrophage exhibits a low minimal inhibition concentration (8 μg/mL Fe-Art M with H2O2 (100 μM)) to combat MRSA and rapidly promote the healing of bacteria-infected wounds on rabbit skin. We suggest that the application of this hedgehog artificial macrophage with “capture and killing” capability and high ROS-catalytic activity will open up a promising pathway to develop antibacterial materials for bionic and non-antibiotic disinfection strategies.

A new phenothiazine derivate is active against Clostridioides difficile and shows low cytotoxicity

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matters of concern for public health. Thioridazine, a compound belonging to the phenothiazine group, has previous shown antimicrobial activity against C. difficile. The purpose of this present study was to investigate the potential of a novel phenothiazine derivative, JBC 1847, as an oral antimicrobial for treatment of intestinal pathogens and CDIs. The minimal inhibition concentration and the minimum bactericidal concentration of JBC 1847 against C. difficile ATCC 43255 were determined 4 μg/mL and high tolerance after oral administration in mice was observed (up to 100 mg/kg bodyweight). Pharmacokinetic modeling was conducted in silico using GastroPlusTM, predicting low (< 10%) systemic uptake after oral exposure and corresponding low Cmax in plasma. Impact on the intestinal bacterial composition after four days of treatment was determined by 16s rRNA MiSeq sequencing and revealed only minor impact on the microbiota in non-clinically affected mice, and there was no difference between colony-forming unit (CFU)/gram fecal material between JBC 1847 and placebo treated mice. The cytotoxicity of the compound was assessed in Caco-2 cell-line assays, in which indication of toxicity was not observed in concentrations up to seven times the minimal bactericidal concentration. In conclusion, the novel phenothiazine derivative demonstrated high antimicrobial activity against C. difficile, had low predicted gastrointestinal absorption, low intestinal (in vitro) cytotoxicity, and only induced minor changes of the healthy microbiota, altogether supporting that JBC 1847 could represent a novel antimicrobial candidate. The clinical importance hereof calls for future experimental studies in CDI models.

Effect of Culture Medium Incorporated with Ocimum sanctum Extract in Enhancing Anti-MRSA activity of Endophytic Fungus, Lasiodiplodia pseudotheobromae

The effect of incorporating plant extract in the culture medium on anti-MRSA activity of Lasiodiplodia pseudotheobromae IBRL OS-64, was investigated in the present study. On disk diffusion assay, the ethyl acetate fungal extract from culture medium supplemented with host plant extract (HPE) of Ocimum sanctum leaves demonstrated good anti-MRSA activity with a diameter inhibition zone of 22.6±0.6 mm. Meanwhile, the minimal inhibition concentration (MIC) values of the extract from YES broth and YES broth incorporated with HPE were 1000 µg/mL and 250 µg/mL, respectively. The MBC values were 8000 µg/mL and 500 µg/mL, respectively. The YES + HPE extracts exerted bactericidal effect against the test bacteria since the MBC/MIC ratio was less than or equal to 4. The time-kill study revealed a 90% of growth reduction of MRSA ATCC 33591 after 16 h exposure to the fungal extract cultured in YES + HPE. Ironically, for fungal extract grown in YES broth, time-kill curve showed a regrowth pattern of bacterial cells after 24 h exposed to the extract. Therefore, the present study suggested that the addition of HPE in the culture medium could enhance the anti-MRSA activity of endophytic fungus, L. pseudotheobromae IBRL OS-64 against MRSA ATCC 33591.

Anti-Quorum Sensing and Antioxidant Activity of Essential Oils Extracted From Juniperus Species, Growing Spontaneously in Tebessa Region (East of Algeria)

The chemical composition of essential oils (EOs) extracted from the aerial parts of 2 species of Juniperus was determined by Gas Chromatography-Mass Spectrometry (GC-MS). In total, 65 and 58 compounds accounting for 90.3% and 89.8% of the whole chemical composition of Juniperus oxycedrus (JO) and Juniperus phoenicea (JP) were identified, respectively, with α-pinene, α-amorphene, terpinen-4-ol, α-terpinene, and β-elemene, as major components. For the first time, the capacity to inhibit quorum-sensing for Chromobacterium violaceum CV026 and CV12472 by the investigated EOs was evaluated. Both oils exhibited good violacein inhibition on CV12472 with 100.0 ± 0.0% inhibition at minimal inhibition concentration (MIC) values. Besides, the quorum-sensing inhibition of CV026 was high at MIC for JO essential oil from fruits (JOF, 16.3 ± 2.0 mm), JO leaves (JOL, 12.5 ± 3.5 mm), JP fruits (JPF, 19.7 ± 2.5 mm), and JP leaves (JPL, 21.1 ± 5.0 mm). On both CV12472 and CV026, essential oil from J. phoenicea leaves was the most active inhibitor. All investigated EOs inhibited swarming motilities in flagellated Pseudomonas aeruginosa (PA01) in a concentration-dependent manner, and those from JP were more active than EOs from JO. Moreover, these EOs showed good antioxidant potential according to DPPH● and FRAP methods, especially the EO from JO leaves with an IC50 DPPH● inhibition value of 20.2 ± 1.0 mg/mL. Based on the obtained results, the investigated EOs are good candidates to combat microbial resistance be used as alternatives to conventional antibiotics, and equally find applications in food biosafety as preservatives.

Antibacterial and Antibiofilm Activities of Novel Antimicrobial Peptides against Multidrug-Resistant Enterotoxigenic Escherichia Coli

Post-weaning diarrhea due to enterotoxigenic Escherichia coli (ETEC) is a common disease of piglets and causes great economic loss for the swine industry. Over the past few decades, decreasing effectiveness of conventional antibiotics has caused serious problems because of the growing emergence of multidrug-resistant (MDR) pathogens. Various studies have indicated that antimicrobial peptides (AMPs) have potential to serve as an alternative to antibiotics owing to rapid killing action and highly selective toxicity. Our previous studies have shown that AMP GW-Q4 and its derivatives possess effective antibacterial activities against the Gram-negative bacteria. Hence, in the current study, we evaluated the antibacterial efficacy of GW-Q4 and its derivatives against MDR ETEC and their minimal inhibition concentration (MIC) values were determined to be around 2~32 μg/mL. Among them, AMP Q4-15a-1 with the second lowest MIC (4 μg/mL) and the highest minimal hemolysis concentration (MHC, 256 μg/mL), thus showing the greatest selectivity (MHC/MIC = 64) was selected for further investigations. Moreover, Q4-15a-1 showed dose-dependent bactericidal activity against MDR ETEC in time–kill curve assays. According to the cellular localization and membrane integrity analyses using confocal microscopy, Q4-15a-1 can rapidly interact with the bacterial surface, disrupt the membrane and enter cytosol in less than 30 min. Minimum biofilm eradication concentration (MBEC) of Q4-15a-1 is 4× MIC (16 μg/mL), indicating that Q4-15a-1 is effective against MDR ETEC biofilm. Besides, we established an MDR ETEC infection model with intestinal porcine epithelial cell-1 (IPEC-1). In this infection model, 32 μg/mL Q4-15a-1 can completely inhibit ETEC adhesion onto IPEC-1. Overall, these results suggested that Q4-15a-1 may be a promising antibacterial candidate for treatment of weaned piglets infected by MDR ETEC.

Effects of Growing Substrate and Nitrogen Fertilization on the Chemical Composition and Bioactive Properties of Centaurea raphanina ssp. mixta (DC.) Runemark

The Mediterranean basin is abundant in wild edible species with numerous health beneficial effects due to the presence of various bioactive phytochemicals. In the present work, the effect of nitrogen fertilization rates (0 ppm, (N0), 200 ppm (N1), 400 ppm (N2), and 600 ppm (N3) of total N) and growth substrate composition (soil or peat/perlite (2/1; v/v)) on the chemical composition and bioactive properties of Centaurea raphanina ssp. mixta plants was evaluated. The results of the study showed that both the tested factors affected nutritional value of the edible leaves, with the soil × N1 treatment being the most beneficial for fat, protein, and carbohydrate content and energetic value. On the other hand, the peat/perlite-grown plants that received 200 ppm of N had the highest content in α-, γ-, and total tocopherols, while the control treatment of soil-grown plants was the richest in individual and total sugars. Oxalic, citric, and total organic acids were the highest in the N2 × soil treatment, while malic acid was the highest in control treatment of the same substrate. The main fatty acids were palmitic, α-linolenic, and linoleic acids, with the highest contents being observed in the N0 × soil, N3 × soil, and N3 × peat/perlite treatments, respectively. The major phenolic compounds were pinocembrim neohesperidoside and pinocembrim acetyl neohesperidoside isomer II, with the highest content being observed in the N1 × soil treatment. The highest antihemolytic activity was observed in the N3 × peat/perlite treatment, while the most effective treatments against lipid peroxidation were N0 (in both soil and peat/perlite combinations) and N1 × peat/perlite. Lastly, all the tested extracts (except for N1 × soil) showed promising cytotoxic effects against HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), and NCI-H460 (non-small-cell lung cancer), while all the tested extracts exhibited better antifungal activities (lower minimal inhibition concentration (MIC) values) against Trichoderma viride than the positive controls. Overall, the present results suggest that the application of cost-effective practices such as the nitrogen application and the selection of growth substrate may regulate the chemical composition and the bioactive properties of C. raphanina ssp. mixta species and increase its added value under commercial cultivation conditions.

Abstract 15682: A Novel Antibiotic Delivery Approach Enhances Salvage of Infected Cardiovascular-implantable Electronic Devices

Introduction: Infection of cardiovascular-implantable electronic devices (CIED) is a serious complication. Systemic antibiotic therapy is considered ineffective, justifying the current guidelines proposing total CEID and lead extraction. Objective: To evaluate the safety and efficacy of continuous in-situ-targeted, ultra-high concentration of antibiotics (CITA) in CIED infections limited to the subcutaneous pocket. Methods: Infected CIED were treated with CITA, delivered to the CIED pocket following minimally invasive surgery (MIS). The CIED were submerged in a 10 2 -10 3 minimal inhibition concentration (MIC) solution of vancomycin and/or gentamicin as per daily conventional dosing. Serum antibiotic levels were regulated by adjusting the concentration and flow of pocket irrigation. CIED salvage rate was assessed. Results: A total of 937 and 481serum assays of vancomycin and gentamicin, respectively, from 83 infected CIED was evaluated (Figure). Target antibiotic serum levels were obtained at 12-48 hours. Median treatment was 8 days (IQR 5,12). Parallel intravenous (IV) vancomycin treatment was applied in 6 (7%) infections. Vancomycin serum levels exceeded a trough limit of 20μg/ml in 20 (2.1%) assays, of which 19/20 received parallel IV treatment. No peak gentamicin levels were recorded. Gentamicin exceeded trough serum level reaching 2-3μg/ml in 80 (16.6%), reached potentially toxic levels of 3-5μg/ml in 33 (6.9%), and exceeded 5μg/ml in 10 (2.1%) assays. CITA resulted in a 1-year salvage of 71 (86%) CIED. Treatment failed in 12 infections, resulting in 8 uneventful total CIED extractions. The 30-day mortality rate was 2.4%. Conclusions: CITA provided ultra-high pocket antibiotic levels, with its safety confirmed by systemic level assays. CITA combined with MIS enabled salvage of 86% of infected CIED. The CITA-MIS approach may serve as an initial therapeutic option prior to the extraction of locally infected CIED, especially in high-risk patients.