Agar Diffusion Assay Research Articles

Electrostatic loading and photoredox-accelerated release of antibiotics from oligoviologen-crosslinked hydrogels using red light

Hydrogel drug delivery is the subject of an ever-expanding industry where delivery occurs by numerous methods, many of them passive in nature. On-command, stimuli-driven release, particularly using light to initiate spatiotemporal, controlled release of cargo is a more attractive approach than passive release methods. Here, we report a novel method for electrostatically loading negatively charged dye and antibiotic cargo in biocompatible, oligoviologen-crosslinked hydrogels equipped with an integrated Aza-boron-dipyrromethene (Aza-BODIPY) photocatalyst. Following loading, the release of the electrostatically bound cargos can be accelerated upon irradiation with red light (660 nm) through a photoinduced electron transfer (PET) process that reduced the oligoviologen subunits. A free radical polymerization method was used to fabricate the hydrogels with a hydroxyethyl acrylate backbone, co-crosslinked by a viologen hexamer (6V-St•12Cl) and Aza-BODIPY derivative. Irradiation of the Aza-BODIPY photocatalyst led to a reduction of the viologen subunits to their monoradical cation oxidation state via PET. The photocatalyst was regenerated in situ in a triethanolamine (TEOA) solution, which served as a sacrificial reductant. Negatively charged dye (i.e., methyl orange, MO) was electrostatically loaded into the hydrogel through counteranion metathesis, followed by irradiation with red light to release the dye as the subunits of the viologen crosslinker lost positive charges (i.e., V2+ to V●+). Controlled release of the dye was tracked by UV/Vis absorption spectroscopy, yielding up to 74 % of cargo released in 75 h. Negatively charged antibiotic cargo (i.e., nalidixic acid) was then electrostatically loaded into and released from hydrogels by the same method. Controlled release of the drug was tracked by agar diffusion and liquid broth microdilution antibacterial susceptibility assays, where bacterial growth inhibition was recorded, confirming the same release trends observed for the dye cargo.

Bioprospecting of secondary metabolites produced by endophytic actinomycete isolated from Aloe vera

The aim of this work to evaluate the antibacterial action of secondary metabolites produced by endophytic actinomycetes, against isolates of medical interest. For such, the bacteria were isolated from the botanical sample and classical morphological identification was performed. The induction of metabolites production was carried out using the Submerged Fermentation technique, followed by purification of the extract. The evaluation of antibacterial activity was accomplished by using agar diffusion and Minimum Inhibitory Concentration (MIC) assays. Macro and microscopic analysis of the grown microorganism allowed it to be identified as belonging to the genus Streptomyces. Test results demonstrated that the extract was able to inhibit the growth of C. propinquum 431024, with an inhibition zone of 27±0 mm and of C. propinquum 36063, with an inhibition zone of 24±0 mm and MIC of 32.1 µg/mL for boths. The evaluation of biofilm formation resulted in: C. propinquun 421024 as a weak producer and C. propinquun 36063 as a moderate producer. The biofilm interference test showed that compared to the control there was a reduction in biofilm formation of 22% for C. propinquun 421024, as well as a reduction in pre-formed biofilm of 27%. There was a 48% reduction in the formation and 42% in the pre-formed biofilm of C. propinquun 36063. Lastly, it was concluded that the secondary metabolites produced by the endophytic actinomycetes demonstrated an antibiotic potential against clinical strains (nasopharynx) of Corynebacterium propinquum, a rare pathogen described for causing infections in patients with pulmonary involvement, pleural effusion, bacteremia and endocarditis and which presents an emerging antibiotic resistance.

Phytochemical profile and antimicrobial activity of essential oils from two Syzygium species against selected oral pathogens

BackgroundThe genus Syzygium (Myrtaceae) comprises several essential oil-rich species that are utilized traditionally for treating tooth infections and toothache. The current study aimed to extract essential oils (EOs) from the leaves of Syzygium samarangense and Syzygium malaccense cultivated in Egypt for the first time and screen their antimicrobial potential against oral-related pathogens.MethodsThe intended EOs were extracted using hydrodistillation (HD) by boiling fresh leaves with distilled water; supercritical fluid (SF) by extracting the dried leaves using supercritical CO2 at 40 °C and 150 bar; and the headspace (HS) in which the fresh leaves were heated in a glass vial and the vaporized aroma were analyzed. The volatile constituents were analyzed using GC/MS and identified by comparing the experimental Kovats' retention indices with the literature. The antimicrobial activity was assessed against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Candida albicans using agar diffusion, microwell dilution, and biofilm formation assays. Statistical significance (p < 0.05) was determined by applying one-way ANOVA and Duncan's post hoc test.ResultsThe yield of the extracted EOs differs between the applied methods, and the SF approach harvested the maximum (0.52–0.46%). The GC–MS analysis of SF EOs revealed a discrepancy between the two species. Since S. malaccense showed an abundance of hydrocarbons represented mainly by squalene (60.60%), S. samarangense was deemed to have oxygenated sesquiterpenes exemplified in globulol (52.09%). On the other side, the HD and HS EOs were sequentially comparable, while differed in the percentage of their majors. γ-terpinene (33.06%) pioneered the HS-derived aroma of S. malaccense, while S. samarangense was abundant with α-pinene (30.18%). Concurrently, the HD EOs of S. malaccense and S. samarangense were commonly denoted by caryophyllene oxide (8.19%-18.48%), p-cymene (16.02%- 19.50%), and γ-terpinene (12.20%-17.84). Ultimately, both species EOs exhibited broad-spectrum antimicrobial potential, although the HD EO was more potent than the SF EO. The HD EOs of both species potently inhibited the growth of E. coli (MIC 3.75 µL/mL) and suppressed C. albicans biofilm formation by 83.43 and 87.27%, respectively. The SF-EOs efficiently suppressed the biofilm formation of Gram-positive bacteria by 76.45%-82.95%.ConclusionEOs extracted from both species by different methods possessed a unique blend of volatile components with broad-spectrum antimicrobial activity. They were promoted as bioactive hits for controlling oral infections, however further investigations concerning their safety in clinical settings are needed.

AgNPs-Modified Polylactic Acid Microneedles: Preparation and In Vivo/In Vitro Antimicrobial Studies.

To prepare polylactic acid microneedles (PLAMNs) with sustained antibacterial effect to avoid skin infection caused by traditional MNs-based biosensors. Silver nanoparticles (AgNPs) were synthesized using an in-situ reduction process with polydopamine (PDA). PLAMNs were fabricated using the hot-melt method. A series of pressure tests and puncture experiments were conducted to confirm the physicochemical properties of PLAMNs. Then AgNPs were modified on the surface of PLAMNs through in-situ reduction of PDA, resulting in the formation of PLAMNs@PDA-AgNPs. The in vitro antibacterial efficacy of PLAMNs@PDA-AgNPs was evaluated using agar diffusion assays and bacterial liquid co-culture approach. Wound healing and simulated long-term application were performed to assess the in vivo antibacterial effectiveness of PLAMNs@PDA-AgNPs. The MNs array comprised 169 tiny needle tips in pyramidal rows. Strength and puncture tests confirmed a 100% puncture success rate for PLAMNs on isolated rat skin and tin foil. SEM analysis revealed the integrity of PLAMNs@PDA-AgNPs with the formation of new surface substances. EDS analysis indicated the presence of silver elements on the surface of PLAMNs@PDA-AgNPs, with a content of 14.44%. Transepidermal water loss (TEWL) testing demonstrated the rapid healing of micro-pores created by PLAMNs@PDA-AgNPs, indicating their safety. Both in vitro and in vivo tests confirmed antibacterial efficacy of PLAMNs@PDA-AgNPs. In conclusion, the sustained antibacterial activity exhibited by PLAMNs@PDA-AgNPs offers a promising solution for addressing skin infections associated with MN applications, especially when compared to traditional MN-based biosensors. This advancement offers significant potential for the field of MN technology.

Unveiling antibacterial and antioxidant activities of zinc phosphate-based nanosheets synthesized by Aspergillus fumigatus and its application in sustainable decolorization of textile wastewater

BackgroundThe development of an environment-friendly nanomaterial with promising antimicrobial and antioxidant properties is highly desirable. The decolorization potentiality of toxic dyes using nanoparticles is a progressively serious worldwide issue.MethodsThe successful biosynthesis of zinc nanoparticles based on phosphates (ZnP-nps) was performed using the extracellular secretions of Aspergillus fumigatus. The antibacterial activity of the biosynthetic ZnP-nps was investigated against Gram-negative bacteria and Gram-positive bacteria using the agar diffusion assay method. The antioxidant property for the biosynthetic nanomaterial was evaluated by DPPH and H2O2 radical scavenging assay.ResultsRemarkable antibacterial and antiradical scavenging activities of ZnP-nps were observed in a dose-dependent manner. The minimum inhibitory concentration (MIC) for Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was 25 µg/ml, however, the MIC for Bacillus subtilis was 12.5 µg/ml. The maximum adsorptive performance of nanomaterial was respectively achieved at initial dye concentration of 200 mg/L and 150 mg/L using methylene blue (MB) and methyl orange (MO), where sorbent dosages were 0.5 g for MB and 0.75 g for MB; pH was 8.0 for MB and 4.0 for MO; temperature was 30 °C; contact time was 120 min. The experimental data was better obeyed with Langmuir’s isotherm and pseudo-second-order kinetic model (R2 > 0.999). The maximum adsorption capacity (qmax) of MB and MO dyes on nanomaterial were 178.25 mg/g and 50.10 mg/g, respectively. The regenerated nanomaterial, respectively, persist > 90% and 60% for MB and MO after 6 successive cycles. The adsorption capacity of the prepared zinc phosphate nanosheets crystal toward MB and MO, in the present study, was comparable/superior with other previously engineered adsorbents.ConclusionsBased on the above results, the biosynthesized ZnP-nanosheets are promising nanomaterial for their application in sustainable dye decolorization processes and they can be employed in controlling different pathogenic bacteria with a potential application as antiradical scavenging agent. Up to our knowledge, this is probably the first study conducted on the green synthesis of ZnP-nanosheets by filamentous fungus and its significant in sustainable dye decolorization.

A novel pyrazolone complex P-FAH-Cu-bpy induces death of Escherichia coli and Staphylococcus aureus by disrupting cell structure and blocking energy.

A novel pyrazolone-based copper complex [Cu(L)(bpy)]∙CH3OH (P-FAH-Cu-bpy) was synthesized and previously characterized to have antitumor properties. This study aimed to investigate its antibacterial properties and action modes against Escherichia coli and Staphylococcus aureus. By agar diffusion assay, P-FAH-Cu-bpy showed strong antibacterial activity against E. coli and S. aureus with the diameter of inhibition zone of 10.17-12.50mm and 11.83-14mm, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the complex were 1.5 and 3μM, respectively. Destroyed bacteria cells and debris were clearly observed by SEM. At 2 MIC and 4 MIC of P-FAH-Cu-bpy, 1.1683 and 1.9083pg copper per cell was taken by E. coli, and 4.5670 and 8.5250pg per cell by S. aureus, respectively. Multi-step resistance selection showed both bacteria were sensitive to P-FAH-Cu-bpy without induction of resistance within 30 generations. With P-FAH-Cu-bpy treatment, the release of nucleotides and proteins and alkaline phosphatase was increased, but the activity of K+-Na+-ATPase and Ca2+-Mg2+-ATPase and membrane conductivity were decreased in both pathogens. In conclusion, P-FAH-Cu-bpy induced death of both bacteria by destroying the cell membrane structure and blocking energy and exhibited strong antibacterial activity against E. coli and S. aureus without inducing microbial resistance.

Efficacy of Lemon Myrtle Essential Oil as a Bio-Fungicide in Inhibiting Citrus Green Mould.

The effectiveness of lemon myrtle (LM) (Backhousia citriodora) essential oil (EO) was investigated to combat Penicillium digitatum by in vitro agar diffusion and vapour assay and in artificially infected oranges. The main constituent of LM EO was revealed as citral when analysed in gas chromatography-mass spectrometry. Pure citral was also included in the experiment for comparison. The in vitro fungal growth was significantly inhibited by LM EO at 1, 2, 3, 4 and 5 μL per disc while complete growth inhibition by both the pure citral and LM EO occurred at 4 and 5 μL per disc. Inoculated fruits treated by dipping in 1000 μL L-1 LM EO solutions for 5, 10, 15, 30 and 120 s showed significantly lower fungal wounds compared to control. While longer dipping times led to some rind injuries, fruits with a 5 and 10 s dip were found free from any injury. The evaluation after dipping and storage confirmed that the fruits maintained the sensory attributes and were not compromised by the incorporation of the essential oil. The results of this study indicate that LM EO can be a promising alternative to synthetic fungicides for preserving the quality of citrus fruits during storage.

Synthesis, docking study and antimicrobial activity evaluation of pyridyl amides of thieno[2,3-d]pyrimidine-4-carboxylic acid

The aim. The combination in one molecule of pharmacophore fragments of thieno[2,3-d]pyrimidine-4-carboxylic acids with the fragments of 2- or 4-aminopyrimidine by peptide coupling promoted acylation in order to develop the new drug-like molecules with antimicrobial activity. Materials and methods. The molecular docking studies were performed with the AutoDock Vina та AutoDockTools 1.5.6 programs; TrmD Pseudomonas aeruginosa PDB ID – 5ZHN was used as the protein target. Synthetic methods of peptide coupling were used. 1H and 13C NMR spectra were recorded with a Varian-400 spectrometer at 400 MHz and Bruker Avance DRX 500 device at 500 MHz and 125 MHz in DMSO-d6 as a solvent, using TMS as the internal standard. LC-MS analysis of the compounds was carried out with Agilent 1100 HPLC з with atmospheric pressure chemical ionization (APCI). The studied derivates were tested in vitro for their antibacterial and anti-fungal activities using agar diffusion and serial dilutions resazurin-based microdilution assays (RBMA). Results and discussion. By the combination of the pharmacophore fragments of thieno[2,3-d]pyrimidine-4-carboxylic acids with the fragments of 2- of 4-aminopyrimidine, the combinatorial library of amides was constructed. For this library of compounds, the potential of antimicrobial activity was revealed using docking studies to the TrmD enzyme isolated from P. aeruginosa. The peptide coupling promoted by 1,1'-carbonyldiimidazole was found to be effective for the synthesis of pyridyl amides of thieno[2,3-d]pyrimidine-4-carboxylic acids, and it allowed to combine these pharmacophores in one molecule. The results of antimicrobial activity study revealed the broad spectrum of antimicrobial activity for N-(pyridin-4-yl)-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine-4-carboxamide (2g), while 5,6-dimethyl-N-(6-methylpyridin-2-yl)thieno[2,3-d]pyrimidine-4-carboxamide (2c) showed the best MIC value against the reference strain of Pseudomonas aeruginosa ATCC 10145. N-(6-Methylpyridin-2-yl)-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine-4-carboxamide (2h) was also found to be active against Pseudomonas aeruginosa. Conclusions. An effective method for the synthesis of pyridyl amides of thieno[2,3-d]pyrimidine-4-carboxylic acid has been developed. The amides molecular docking method showed their ability to inhibit TrmD enzyme isolated from P. aeruginosa; the further in vitro studies of the compounds showed the rationality of the further studies of the derivatives with 2-amino-6-methylpyridine in amide substituent because this fragment favoured the selectivity against Pseudomonas aeruginosa

Computational and Experimental Investigation of Antibacterial Properties of Some Fluorinated Thioureas

Herein, fluorinated thioureas with various substituents (F1-F3) were synthesized and characterized spectroscopically and analytically to investigate their properties as antibacterial agents. Prior to experimental studies, Density Functional Theory (DFT) modeling was performed at B3LYP/6-31G (d,p) to complement and offer comparative overview with the experimental findings. With regards to all theoretical analyses, the Frontier Molecular Orbital (FMO) demonstrated a desirable low HOMO-LUMO gap for all three designated derivatives, particularly F1 (1.60 eV), which is consistent with its GCRDs values, namely its high electronegativity and low hardness values that corresponds to low LUMO energy, suggesting higher bacterial activity. Agar diffusion assay was used for the preliminary screening of in-vitro antibacterial activity against pathogenic Gram-positive and Gram-negative bacteria. All three derivates successfully inhibit at least two bacterial strains (B. cereus, S. aureus), but none were able to penetrate into E. coli. The highest inhibition rate was exhibited by F3 at 33.33 ± 0.71% against S. aureus, whilst F1 only managed to gain 29.63 ± 0.00% of inhibition rate for the same bacterium. Meanwhile, the lowest penetration rate was recorded at 1.53 ± 0.35% by F2 against B. cereus.

Enhancing the Functional Properties of Tea Tree Oil: In Vitro Antimicrobial Activity and Microencapsulation Strategy.

In the context of addressing antimicrobial drug resistance in periocular infections, Tea Tree Oil (TTO) has emerged as a promising therapeutic option. This study aimed to assess the efficacy of TTO against bacterial strains isolated from ocular infections, with a particular focus on its ability to inhibit biofilm formation. Additionally, we designed and analyzed microcapsules containing TTO to overcome certain unfavorable physicochemical properties and enhance its inherent biological attributes. The quality of TTO was confirmed through rigorous analysis using GC-MS and UV-Vis techniques. Our agar diffusion assay demonstrated the effectiveness of Tea Tree Oil (TTO) against ocular bacterial strains, including Corynebacterium spp., coagulase-negative Staphylococcus spp., and Staphylococcus aureus, as well as a reference strain of Staphylococcus aureus (ATCC 25923). Notably, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for all tested microorganisms were found to be 0.2% and 0.4%, respectively, with the exception of Corynebacterium spp., which exhibited resistance to TTO. Furthermore, TTO exhibited a substantial reduction in biofilm biomass, ranging from 30% to 70%, as determined by the MTT method. Through the spray-drying technique, we successfully prepared two TTO-containing formulations with high encapsulation yields (80-85%), microencapsulation efficiency (90-95%), and embedding rates (approximately 40%). These formulations yielded microcapsules with diameters of 6-12 μm, as determined by laser scattering particle size distribution analysis, and exhibited regular, spherical morphologies under scanning electron microscopy. Importantly, UV-Vis analysis post-encapsulation confirmed the presence of TTO within the capsules, with preserved antioxidant and antimicrobial activities. In summary, our findings underscore the substantial therapeutic potential of TTO and its microcapsules for treating ocular infections.

Lactiplantibacillus plantarum Postbiotics Suppress Salmonella Infection via Modulating Bacterial Pathogenicity, Autophagy and Inflammasome in Mice.

Our study aimed to explore the effects of postbiotics on protecting against Salmonella infection in mice and clarify the underlying mechanisms. Eighty 5-week-old C57BL/6 mice were gavaged daily with Lactiplantibacillus plantarum (LP)-derived postbiotics (heat-killed bacteria, LPBinactive; culture supernatant, LPC) or the active bacteria (LPBactive), and gavaged with Salmonella enterica Typhimurium (ST). The Turbidimetry test and agar diffusion assay indicated that LPC directly inhibited Salmonella growth. Real-time PCR and biofilm inhibition assay showed that LPC had a strong ability in suppressing Salmonella pathogenicity by reducing virulence genes (SopE, SopB, InvA, InvF, SipB, HilA, SipA and SopD2), pili genes (FilF, SefA, LpfA, FimF), flagellum genes (FlhD, FliC, FliD) and biofilm formation. LP postbiotics were more effective than LP on attenuating ST-induced intestinal damage in mice, as indicated by increasing villus/crypt ratio and increasing the expression levels of tight junction proteins (Occludin and Claudin-1). Elisa assay showed that LP postbiotics significantly reduced ST-induced inflammation by regulating the levels of inflammatory cytokines (the increased IL-4 and IL-10 and the decreased TNF-α) in serum and ileum (p < 0.05). Furthermore, LP postbiotics inhibited the activation of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome by decreasing the protein expression of NLRP3 and Caspase-1, and the gene expression of Caspase-1, IL-1β and IL-18. Meanwhile, both LPC and LPB observably activated autophagy under ST infection, as indicated by the up-regulated expression of LC3 and Beclin1 and the downregulated p62 level (p < 0.05). Finally, we found that LP postbiotics could trigger an AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy. In summary, Lactiplantibacillus plantarum-derived postbiotics alleviated Salmonella infection via modulating bacterial pathogenicity, autophagy and NLRP3 inflammasome in mice. Our results confirmed the effectiveness of postbiotics agents in the control of Salmonella infection.

Food-protecting films based on soy protein isolate and natural deep eutectic solvents: Antimicrobial and antioxidant properties

Natural deep eutectic solvents (NADES) have just recently emerged as promising plasticizers in food coating applications owing to their excellent anti-frosting properties, low cost, and biosafety. Herein, we report for the first time the use of NADES as bioactive additives of soy protein isolate (SPI) to fabricate functional packaging materials. A neoteric eutectic solvent based on choline chloride/tannic acid (ChCl/TA) and the classical ChCl/citric acid mixture (ChCl/Cit) were explored to be integrated into the protein matrix at really high ratios (150 and 200 wt%). The resulting films were characterized by assessing morphological, physicochemical, and mechanical properties, as well as antioxidant and antimicrobial activities. The films exhibited a hydrophilic surface but limited water swelling (less than 10%), suggesting the formation of a crosslinked network between NADES and SPI. The ChCl/Cit films demonstrated potent antimicrobial activity in bacterial growth inhibition assay on chicken breast as well as in agar diffusion assays with inhibition zone diameters over 20 mm for gram+ and gram-bacteria, while ChCl/TA films showed the highest tensile strength (ranging from 744 to 2740 kPa), superior antioxidant activity (DPPH scavenging activity of ≈82%), and high light barrier properties, advantageous for storing light-sensitive food products. The incorporation of high concentrations of NADES in film production provides a novel and practical approach to improving the properties of protein materials for food packaging applications.

Room temperature magnetic ordering and in vitro antibacterial potency of Nd3+ doped CeO2 against the pathogenic bacterial strains

In this study, 5% Nd3+ doped Cerium oxide (Ce0.95Nd0.05O2) was prepared by a modified solid-state reaction method. Crystallite size and lattice strain were determined from the Williamson-Hall (W-H) plot. The four distinct peaks in the photoluminescence (PL) spectra in the visible region signify the structural defects within the crystal lattice. Room temperature ferromagnetism (RTFM) was observed from the hysteresis loop, and the mathematical fitting of the hysteresis loop suggests ferromagnetic dominance (∼83%) in the magnetic response. The virgin M-H loop was fitted by the ferromagnetic Brillouin function, which suggests possible domain formation in the sample. The in vitro antibacterial activity of 5% Nd3+doped CeO2 was evaluated against two clinically isolated multidrug-resistant pathogenic bacterial strains, namely MLD 2 (Escherichia coli) and MLD 4 (Staphylococcus aureus), utilizing the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), disc agar diffusion (DAD) assays, etc. Notably, compared to the MLD 4 bacterial strain, the synthesized material revealed comparable better antibacterial results against the Gram-negative MLD 2 and in comparison, to the MLD 2 bacterial strain, a relatively higher concentration of the sample is required to completely eradicate the bacterial population of MLD 4. This implies that Ce0.95Nd0.05O2 can destroy the growth of the two tested bacterial strains. In the context of cytotoxicity, up to a certain concentration, the synthesized particle demonstrated no appreciable toxicity towards healthy PBMCs.

Synergistic action of endophytic fungi secondary metabolites against multidrug-resistant strains of Staphylococcus aureus

The global threat to treatment of infections associated with multidrug-resistant microorganisms as well as side effects associated with combination therapy calls for the urgent development of new medicines to address these challenges. Endophytic fungi are efficient bio synthesizers and offer a wide array of these reliable bioactive compounds. The present study is aimed at evaluating the effect of combined extracts of endophytic fungi isolated from the leaves of Musa paradisiaca against multidrug-resistant Staphylococcus aureus (MRSA) strains. Screening for multidrug resistance was carried out using the Kirby-Bauer disc diffusion assay followed by a preliminary assay of endophytic fungal extracts bioactivity using agar diffusion assay against selected MRSA. A synergistic study using checkerboard assay technique was performed, then extracts were subjected to quantitative evaluation of secondary metabolites using Gas Chromatography with Flame Ionization Detector. Axenic cultures of five endophytic fungi from M. paradisiaca (EMp1-Emp5) were isolated from the sampled leaves. All the endophytic fungal extracts inhibited the growth of all the multi-drug resistant Staphylococcus aureus at 0.5 mg/mL. Varying inhibition zones that were concentration-dependent were observed. The combined effects of EMp3 and EMp5 extracts at 9:1; 8:2; 4:6; 3:7; and 2:8 combination ratios produced synergistic effects against S. aureus (S18) with very low Fractional inhibitory Concentrations of 0.03:0.01, 0.01:0.01, 0.006:0.02, 0.01:0.04 and 0.01:0.1 mg/mL respectively. Several bioactive secondary metabolites such as anthocyanin, (-) epicatechin, naringenin, resveratrol, catechin, rutin, phenol were produced by the fungal endophytes. Our findings further affirm fungal endophytes associated with M. paradisiaca leaves as producers of natural antibacterial agents with synergistic potentials.

Screening and Functional Analyses of Novel Cecropins from Insect Transcriptome.

Antibiotic resistance is a significant and growing threat to global public health. However, antimicrobial peptides (AMPs) have shown promise as they exhibit a broad spectrum of antibacterial activities with low potential for resistance development. Insects, which inhabit a wide range of environments and are incredibly diverse, remain largely unexplored as a source of novel AMPs. To address this, we conducted a screening of the representative transcriptomes from the 1000 Insect Transcriptome Evolution (1KITE) dataset, focusing on the homologous reference genes of Cecropins, the first identified AMPs in insects known for its high efficiency. Our analysis identified 108 Cecropin genes from 105 insect transcriptomes, covering all major hexapod lineages. We validated the gene sequences and synthesized mature peptides for three identified Cecropin genes. Through minimal inhibition concentration and agar diffusion assays, we confirmed that these peptides exhibited antimicrobial activities against Gram-negative bacteria. Similar to the known Cecropin, the three Cecropins adopted an alpha-helical conformation in membrane-like environments, efficiently disrupting bacterial membranes through permeabilization. Importantly, none of the three Cecropins demonstrated cytotoxicity in erythrocyte hemolysis tests, suggesting their safety in real-world applications. Overall, this newly developed methodology provides a high-throughput bioinformatic pipeline for the discovery of AMP, taking advantage of the expanding genomic resources available for diverse organisms.

Detection and evaluation of the antimicrobial activity of Micrococcin P1 isolated from commensal and environmental staphylococcal isolates against MRSA

Bacteriocins (of different origins) have been proposed as promising alternatives to face antimicrobial resistance-associated health problems. Isolates of the Staphylococcus genus are well-known bacteriocin producers, especially coagulase-negative species. Twenty-eight bacteriocin-producing staphylococcal isolates were selected from a previous study for in-depth characterisation. The antimicrobial activities (AA) of the producing isolates were studied by the spot-on-lawn method and their crude cell-free supernatants (CFS) and butanol extracts (BT) were evaluated by agar diffusion assays against six indicator bacteria, including multidrug-resistant and zoonotic isolates (such as Listeria monocytogenes or methicillin-resistant Staphylococcus aureus [MRSA]). Six bacteriocin-producing isolates showed AA in their CFS, whereas all staphylococcal BT extracts inhibited at least one of the tested indicator bacteria. Micrococcin P1 (MP1) bacteriocin was detected by mass spectrometry in four producing isolates: Staphylococcus aureus-C5802, Staphylococcus hominis-C5835, Staphylococcus sciuri-X3041, and -X3011. Growth curves performed with CFS and BT extracts of the four MP1 producers revealed a strong AA profile against MRSA and Listeria monocytogenes, even when considerably diluted. Moreover, synergism between the BT extract of MP1-producing Staphylococcus sciuri-X3041 and several antibiotics against an MRSA indicator was observed: BT-clindamycin (> 80%) and BT-oxacillin (30%) combinations. For the BT-chloramphenicol combination, synergism and near synergism values were observed in 37% of the combinations. Competition studies revealed potent inhibitory effects of the MP1-producing isolates against the MRSA indicator. These results help to identify Staphylococcus isolates or their bacteriocins as interesting candidates for potential future applications.

Cleistocalyx nervosum var. paniala Extract: Anti-acne, Anti-oxidant, Anti-inflammatory Activities and Safety for Cosmeceutical Applications

This research aimed to investigate the biological activities and safety of extracts from Cleistocalyx nervosum var. paniala for anti-acne cosmeceutical and to determine total phenolic, flavonoid and anthocyanin contents and HPLC fingerprint. The extracts named as whole fruit extract (WF) and seed extract (SE), were macerated with 95% ethanol. Antibacterial effects against Cutibacterium acnes and Staphylococcus aureus were evaluated using agar diffusion, broth microdilution and time-kill kinetic assays. The results revealed that SE presented stronger antibacterial activities against C. acnes and S. aureus with inhibition zone of 22.74 ± 0.52 and 12.67 ± 0.15 mm, respectively. However, the MIC and MBC values were with 6.25 and 3.12 mg/ml for C. acnes and S. aureus, respectively, with a kill time of 2 and 4 h, respectively. SE showed higher phenolic and flavonoid contents compared with WF. In contrast, WF revealed higher anthocyanin content. According to HPLC analysis, ellagic acid was used as a marker. Additionally, anti-oxidant activity of the extracts was investigated by DPPH assay and inhibition on nitric oxide radicals. SE showed better activities with IC50 of 7.51 ± 0.40 and 25.47 ± 1.09 µg/ml, respectively. Therefore, SE was selected for testing with nitric oxide radical producing on RAW 264.7 cell lines which presented 22.99% inhibition. Moreover, SE showed no irritation on Hen's Egg Test Chorioallantoic Membrane. To conclude, SE from C. nervosum exerted stronger biological activities than WF. It also demonstrated a good potential concerning antibacterial on acne-related microorganisms, anti-oxidants, and anti-inflammatory activities. Therefore, it could be a promising bio-active compound to further develop into anti-acne cosmeceutical products. Keywords: Cleistocalyx nervosum, Acne vulgaris, Cutibacterium acnes, Anti-inflammatory activity

Elaboration and characterization of ε-polylysine‑sodium alginate nanoparticles for sustained antimicrobial activity

The ε-polylysine (ε-PL) is a food-grade antimicrobial substance. The cationic ε-PL molecules may interact with anionic components of food matrix causing turbidity, sedimentation, and hampering the antimicrobial activity. Herein, sodium alginate (SA) was used as wall material to encapsulate ε-PL, thereby to synthesize ε-PL-SA nanoparticles (ε-PL-SA-NPs). Monosaccharide composition and molecular weight of SA were characterized. The synthetic scheme is optimized and physicochemical characteristics and antimicrobial potential was investigated. Findings indicate that SA primarily consisted of mannuronic acid (95.25 %), weight average molecular weight (Mw) of SA was 176.464 kDa, and the molecular configuration of SA was irregular line clusters. The encapsulation efficiency (EE) of ε-PL in ε-PL-SA-NPs made under optimum strategy (at pH 6.0, mass ratio of ε-PL to SA is 0.14, and SA concentration is 6 mg/mL) is about 99.74 %. The particle size of ε-PL-SA-NPs is ∼541.86 nm. The SEM image showed that the ε-PL-SA-NPs had a nearly spherical morphology. Zeta-potential and FTIR data reveal the interaction between ε-PL and SA was electrostatic and the hydrogen bonding. Agar diffusion assay exhibit that ε-PL-SA-NPs had antimicrobial activity against Escherichia coli and Staphylococcus aureus. The salmon preservation experiments reveal sustained antimicrobial efficacy of ε-PL-SA-NPs.

A Novel Direct-Fed Microbial for Beef Cattle Has a Supportive Effect against Clostridium perfringens In Vitro and In Vivo

Two experiments were conducted to evaluate the in vitro and in vivo effects of a novel direct-fed microbial (DFM) containing Lactobacillus animalis LA-51, Propionibacterium freudenreichii PF-24, Bacillus licheniformis CH-200, and Bacillus subtilis King (BOVAMINE DEFEND® Plus) against Clostridium perfringens pathogenic strains. In Experiment 1 (in vitro), an agar diffusion assay was performed to qualitatively evaluate the in vitro inhibitory effects of the DFM against C. perfringens types A and C. Including the DFM in the tested yielded inhibition zones with greater than three ring diameters in a 96-well plate. In Experiment 2 (in vivo), twenty 1-day-old beef calves were allocated to control (n = 10) or DFM (n = 10) for 21 days. All calves were orally challenged with 1.0 × 108 colony forming units of C. perfringens type A strain S-107 per head. The procedures such as general health scores, body weight, and fecal sample collections were performed following the C. perfringens challenge. Daily feeding of DFM significantly reduced the incidence of diarrhea while improving general impression and appearance scores of calves. Overall, these results highlight the ability of the DFM containing L. animalis LA-51, P. freudenreichii PF-24, B. licheniformis CH-200, and B. subtilis (BOVAMINE DEFEND® Plus) to inhibit C. perfringens types A and C under different experimental settings.

Preparation and characterization of drug-loaded, electrospun nanofiber mats formulated with zein or zein-based mixtures for wound healing applications.

Electrospun zein mats are known to have poor mechanical and water uptake properties limiting their usefulness as wound dressings.&#x0D; In this study, the effects of the solvent system used, the incorporation of the polymeric additives; polyethylene glycol 20000 (PEG20K) and polyvinylpyrrolidone K30 (PVPK30); and the crosslinking agent Tannic Acid (TA) on the mechanical and water uptake characteristics of zein – based nanofiber mats were investigated.&#x0D; The incorporation of either PEG20K or PVPK30 resulted in an improvement in water vapor sorption and a reduction in water contact-angle of the nanofiber mats. In addition, the incorporation of PEG20K and PVPK30 reduced the ultimate tensile strength, and Young’s modulus while increasing the percent elongation of the nanofiber mats. The use of tannic acid as a crosslinking agent led to an increase in the water vapor sorption, ultimate tensile strength, and Young’s modulus of the nanofiber mats.&#x0D; Mats with smaller average fiber diameter, greater ultimate tensile strength, higher Young's modulus, and greater water vapor sorption were obtained when using 80% (v/v) aqueous ethanol as a solvent system during the preparation of the nanofiber mats when compared to those produced using 60% (v/v) aqueous ethanol. In addition, using solutions with lower zein concentration resulted in mats with lower average fiber diameter, lower ultimate tensile strength and Young’s modulus, and higher percent elongation.&#x0D; Selected formulations were loaded with tetracycline hydrochloride and drug release was evaluated in bulk liquid and using Franz diffusion cells. The use of Franz diffusion cells allowed the discrimination between formulation performance as a function of composition and water uptake properties. Drug release from nanofiber mats was also confirmed by observing the formation of an inhibition zone in cultures of E. coli and S. aureus using the agar diffusion assay.&#x0D; Improved performance of zein nanofiber mats was achieved using polymeric modifiers and crosslinking with tannic acid improving their suitability for wound dressing applications.