Confocal Laser Scanning Microscopy Analysis Research Articles

Ephedra foeminea as a Novel Source of Antimicrobial and Anti-Biofilm Compounds to Fight Multidrug Resistance Phenotype.

Plants are considered a wealthy resource of novel natural drugs effective in the treatment of multidrug-resistant infections. Here, a bioguided purification of Ephedra foeminea extracts was performed to identify bioactive compounds. The determination of antimicrobial properties was achieved by broth microdilution assays to evaluate minimal inhibitory concentration (MIC) values and by crystal violet staining and confocal laser scanning microscopy analyses (CLSM) to investigate the antibiofilm capacity of the isolated compounds. Assays were performed on a panel of three gram-positive and three gram-negative bacterial strains. Six compounds were isolated from E. foeminea extracts for the first time. They were identified by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analyses as the well-known monoterpenoid phenols carvacrol and thymol and as four acylated kaempferol glycosides. Among them, the compound kaempferol-3-O-α-L-(2″,4″-di-E-p-coumaroyl)-rhamnopyranoside was found to be endowed with strong antibacterial properties and significant antibiofilm activity against S. aureus bacterial strains. Moreover, molecular docking studies on this compound suggested that the antibacterial activity of the tested ligand against S. aureus strains might be correlated to the inhibition of Sortase A and/or of tyrosyl tRNA synthase. Collectively, the results achieved open interesting perspectives to kaempferol-3-O-α-L-(2″,4″-di-E-p-coumaroyl)-rhamnopyranoside applicability in different fields, such as biomedical applications and biotechnological purposes such as food preservation and active packaging.

Improved gelling and emulsifying properties of myofibrillar protein from frozen shrimp (Litopenaeus vannamei) by high-intensity ultrasound

The deterioration of shrimp protein functionality during chilling and frozen storage has recently attracted wide attention due to its adverse effects on shrimp quality. The high-intensity ultrasound (20 kHz, 400 W) was applied to improve the protein structure and functional properties of frozen shrimp ( Litopenaeus vannamei ) in this study. Fourier transform infrared (FTIR) spectroscopy showed that the cavitation effect of ultrasound could change the secondary structure of myofibrillar protein (MP) by turning the α-helix and random coil to β-sheet and β-turn. The unfolding of MP conformation increased the reactive sulfhydryl content and surface hydrophobicity accordingly. The structure changes eventually led to the significant improvement of protein functionality. Gel properties analysis showed after ultrasonic treatment, the gel strength and water holding capacity of protein gels were increased gradually. Scanning electron microscopy (SEM) images also demonstrated that ultrasound treatment contributed to the formation of stable network structures in protein gels. In addition, ultrasonic homogenization could increase the emulsifying ability (45.54 m 2 /g to 78.82 m 2 /g) and stability of protein in a short time, which might be related to the decrease of particle size. Confocal laser scanning microscope (CLSM) analysis further illustrated that ultrasound treatment made the emulsion distribution more uniform. The application of these findings will improve the deep processing ability and expand the commercialization of frozen shrimp products. • Ultrasonic treatment led to the unfolding of shrimp MP conformation. • Conformational change led to the increase of SH groups and surface hydrophobicity. • Ultrasound increased gel strength and water holding capacity of shrimp MP gel. • Ultrasound increased the emulsifying capacity and stability of frozen shrimp MP. • CLSM analysis illustrated ultrasound made emulsion distribution more uniform.

Effect of rice bran on properties of yogurt: Comparison between addition of bran before fermentation and after fermentation

Recently, fortifying yogurt with dietary fiber has attracted considerable interest. Since fermentation by lactic acid bacteria might alter the fiber composition and structure, adding fiber into yogurt before and after fermentation might have different effects on properties of yogurt. Therefore, this work investigated effects of adding rice bran before and after fermentation on properties of yogurt to choose a better approach of fortifying yogurt with dietary fiber. Adding rice bran before fermentation significantly decreased syneresis of yogurt at the content of 2.0% and 3.0%, while adding rice bran after fermentation increased syneresis of yogurt. Scanning electron microscopy and confocal laser scanning microscopy analysis indicated that the yogurt gel with rice bran added before fermentation was more compact and porous than the yogurt gel with rice bran added after fermentation. Moreover, the autofluorescence from rice bran added after fermentation was significantly stronger than that in rice bran added before fermentation. Compared with yogurt added with rice bran after fermentation, yogurt added with rice bran before fermentation had higher free p-hydroxybenzoic acid, p-coumaric acid, ferulic acid and sinapic acid contents. These results suggested that fermentation converted bound phenolic acids of rice bran into free ones, which was beneficial to improve the yogurt stability. Thus, the pH, texture and rheological properties of yogurt added with rice bran before fermentation were investigated. It was found that rice bran increased the pH and decreased firmness, consistency, cohesiveness, viscosity and gel strength of yogurt.

4-Allyl-2-methoxyphenol modulates the expression of genes involved in efflux pump, biofilm formation and sterol biosynthesis in azole resistant Aspergillus fumigatus.

Antifungal therapy for aspergillosis is becoming problematic because of the toxicity of currently available drugs, biofilm formation on host surface, and increasing prevalence of azole resistance in Aspergillus fumigatus. Plants are rich source of bioactive molecules and antimicrobial activity of aromatic bioactive compounds draws attention because of its promising biological properties. The present study elucidated the antibiofilm activity of 4-allyl-2-methoxyphenol (eugenol) against azole-resistant environmental A.fumigatus isolates. Soil samples were collected from agricultural fields across India; azole-resistant A. fumigatus (ARAF) were isolated followed by their molecular identification. Antibiofilm activity of eugenol was calculated via tetrazolium based-MTT assay. The expression of the multidrug efflux pumps genes MDR1, MDR4, transporters of the MFS gene, erg11A gene encoding 14α demethylase, and transcription regulatory genes, MedA, SomA and SrbA, involved in biofilm formation of A.fumigatus were calculated by quantitative real time PCR. Out of 89 A.fumigatus isolates, 10 were identified as azole resistant. Eugenol exhibited antibiofilm activity against ARAF isolates, ranging from 312 to 500 µg/mL. Confocal laser scanning microscopy analysis revealed absence of extracellular matrix of ARAF biofilm after eugenol treatment. The gene expression indicated significantly low expression of efflux pumps genes MDR1, MDR4, erg11A and MedA in eugenol treated ARAF isolates when compared with untreated isolates. Our results demonstrate that eugenol effects the expression of efflux pump and biofilm associated genes as well as inhibits biofilm formation in azole resistant isolates of A. fumigatus.

Evaluation of machine-assisted irrigation on removal of intracanal biofilm and extrusion of sodium hypochlorite using a three-dimensionally printed root canal model.

This study aimed to compare the biofilm removal and apical extrusion of sodium hypochlorite (NaOCl) following machine-assisted irrigation using a three-dimensionally (3D) printed dentin-insert model. Multispecies biofilms were formed in a 3D-printed curved root canal model with dentin insert. The model was then placed in a container that was filled with 0.2% agarose gel containing 0.1% m-Cresol purple. Root canals were irrigated with 1% NaOCl using syringe irrigation, sonically agitated (EndoActivator or EDDY) or ultrasonically activated (Endosonic Blue) irrigation. Samples were photographed and the color-changed area was measured. Biofilm removal was assessed using colony-forming unit counting, confocal laser scanning microscopic analysis and scanning electron microscopic observations. The data were analyzed using one-way ANOVA, followed by Tukey test (P < 0.05). EDDY and Endosonic Blue demonstrated significantly greater reduction of biofilms compared to other groups. No significant differences were observed in the remaining biofilm volume in syringe irrigation and EndoActivator groups. Furthermore, EDDY and Endosonic Blue presented with numerous exposed dentinal tubules. EDDY showed significantly greater NaOCl extrusion compared to other groups. Ultrasonic activation with a small-sized nickel-titanium file irrigation system may be beneficial in intracanal biofilm removal avoiding extrusion of NaOCl beyond the root apex.

Combined effect of apigenin and reduced graphene oxide against Enterococcus faecalis biofilms.

Enterococcus faecalis (E. faecalis) is one of the major microorganisms that causes failure of endodontic treatment. This study aimed to investigate the antibacterial activity of apigenin and its synergistic effect with reduced graphene oxide (RGO) in treating E. faecalis biofilms. The antibacterial activities were characterized by viability analysis including colony forming units and confocal laser scanning microscopy (CLSM) analyses. The effect on biofilm biomass was measured using a crystal violet staining method. Live and dead bacteria bio-volumes were determined by CLSM analysis, and the morphology of E. faecalis biofilm after treatment with apigenin and apigenin + RGO was observed by scanning electron microscopy (SEM). The viability of E. faecalis in biofilms decreased by apigenin treatment in a dose-dependent manner. While apigenin alone did not significantly affect the biofilm biomass, apigenin + RGO reduced the biomass in an apigenin concentration-dependent manner. Likewise, the bio-volume of live bacteria decreased and the bio-volume of dead bacteria increased in apigenin-treated biofilms. According to SEM images, apigenin + RGO-treated samples showed less E. faecalis in biofilms than apigenin-only treated samples. The results suggested that the combined use of apigenin and RGO could be a potential strategy for effective endodontic disinfection.

Comparison of the effects of negative pressure wound therapy and negative pressure wound therapy with instillation on wound healing in a porcine model.

Negative pressure wound therapy with instillation (NPWTi) is a novel method based on standard negative pressure wound therapy (NPWT). This study aimed to compare the effects of standard NPWT and NPWTi on bioburden and wound healing in a Staphylococcus aureus (S.aureus) infected porcine model. Green fluorescent protein-labeled S.aureus infected wounds were created on the back of porcine. Wounds were treated with NPWT or NPWT with instillation (saline). The tissue specimens were harvested on days 0 (12 h after bacterial inoculation), 2, 4, 6, and 8 at the center of wound beds. Viable bacterial counts, laser scanning confocal microscopy, PCR, western blot, and histological analysis were performed to assess virulence and wound healing. The bacterial count in the NPWTi group was lower than that of the NPWT group and the difference was statistically significant on day 2, day 4, day 6, and day 8 (P < 0.05). The expression levels of agrA, Eap, Spa, and Hla genes of the NPWTi group were significantly lower than that of the NPWT group on day 8 (P < 0.05). The bacterial invasion depth of the NPWTi group was significantly lower than that of the NPWT group on day 2, day 4, day 6, and day 8 (P < 0.05). Though the NPWTi group showed a significantly increased expression of bFGF and VEGF than that of the NPWT group in the early time (P < 0.05), NPWTi cannot lead to better histologic parameters than the NPWT group (P > 0.05). Our results demonstrated that NPWTi induced a better decrease in bacterial burden and virulence compared with standard NPWT. These advantages did not result in better histologic parameters on the porcine wound model.

Evaluation of the effect of biofilm formation on the reductive transformation of triclosan in cathode-modified electrolytic systems

The performance of electrochemical reduction is often enhanced by electrode modification techniques. However, there is a risk of microbial colonization on the electrode surface to form biofilms in the treatment of actual wastewater with modified electrodes. In this work, the effects of biofilm formation on modified electrodes with reduced graphene oxide (rGO), platinum/carbon (Pt/C), and carbon nanotube (CNT) were investigated in triclosan (TCS) degradation. With biofilm formation, the TCS degradation efficiencies of carbon cloth (CC), rGO@CC, Pt/C@CC, and CNT@CC decayed to 54.53 %, 59.77 %, 69.19 %, and 53.97 %, respectively, compared to the raw electrodes. Confocal laser scanning microscopy and microbial community analysis revealed that the difference in biofilm thickness and activity were the major influencing factors on the discrepant TCS degradation rather than the microbial community structure. The electrochemical performance tests showed that the biofilm formation increased the ohmic resistance by an order of magnitude in rGO@CC, Pt/C@CC, and CNT@CC, and the charge transfer resistance was increased by 2.45, 3.78, and 7.75 times, respectively. The dechlorination and hydrolysis governed the TCS degradation pathway in all electrolysis systems, and the toxicity of electrochemical reductive products was significantly decreased according to the Toxicity Estimation Software Tool analysis. This study presented a systematic assessment of the biofilm formation on modified electrodes in TCS reduction, and the undisputed experimental outcomes were obtained to enrich the knowledge of implementing modified electrodes for practical applications.

Microbiological Properties and Cytotoxicity of PNVCL Hydrogels Containing Flavonoids as Intracanal Medication for Endodontic Therapy.

This study aimed to evaluate the cytotoxicity and microbiological properties of poly (N-vinylcaprolactam)-PNVCL hydrogels containing flavonoids as intracanal medication for endodontic therapy. Antimicrobial activity of ampelopsin (AMP), isoquercitrin and rutin was determined against Enterococcus faecalis, Actinomyces israelii, Lactobacillus casei, Streptococcus mutans, and Fusobacterium nucleatum by the microdilution method. After synthesis and characterization by rheology, PNVCL hydrogels were loaded with AMP and controls calcium hydroxide (CH) and chlorhexidine (CHX), and determined the compounds release profile. PNVCL+AMP, PNVCL+CH, PNVCL+CHX were evaluated on multi-species biofilms and analyzed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). Cytotoxicity was determined after fibroblasts exposure to serial dilutions of AMP and PNVCL hydrogel extracts. AMP was effective against all of the bacteria tested, especially against S. mutans, A. israelli and F. nucleatum. SEM and CLSM analysis showed that PNVCL + AMP caused a significant decrease and disorganization of multi-species biofilms and reduction of intracanal viable cells, superior to the other groups. AMP affected fibroblast viability at concentrations above 0.125 mg/mL, and extracts of PNVCL+AMP showed low cytotoxicity. In conclusion, PNVCL containing AMP demonstrated cytocompatibility and potent effect against multi-species biofilms and could be potential intracanal medication for endodontic purposes.

Microbial interaction energy and EPS composition influenced ultrafiltration membrane biofouling and the role of UV pretreatment

Biofouling is still an important factor limiting ultrafiltration further application, and previous mechanism analyses were mainly focused on extracellular polymeric substances (EPS) composition and concentration. In this study, both biofouling and alleviation mechanisms were further investigated in detail with three typical microorganisms, particularly toward the microbial interaction energy. The results showed that a stronger fouling degree was induced by Pseudomonas putida (rod-shaped) than that induced by Staphylococcus aureus and Enterococcus faecalis (sphere). The computational fluid dynamics results suggested that the larger membrane filtration resistance caused by P. putida was due to more EPS secretion. EPS composition and confocal laser scanning microscopy analysis indicated that polysaccharide in EPS played greater roles on the biofouling than protein. According to the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory, higher interaction energy was induced because of higher content of polysaccharide (e.g., hydroxyl groups) and protein (e.g., aggregated strands of amide I band) in EPS from P. putida, resulting in a thicker biofilm and stronger biofouling degree than that caused by S. aureus and E. faecalis. In addition, both EPS secretion and microbial interaction energy were inhibited by UV pretreatment, resulting in fouling alleviated. This work will be beneficial for further biofouling control in actual water treatment plants.

Putative inactivation mechanism and germicidal efficacy of induced electric field against Staphylococcus aureus

Induced electric field (IEF), as an alternative non-conventional processing technique, is utilized to sterilize liquid foods. In this study, the survival and sublethal injury of S. aureus under IEF were investigated in 0.85% normal saline, and the inactivation mechanism of IEF was expounded. The plate count results showed that the sublethal injury rates remained above 90% after IEF treatment for more than 8.4 s, and 7.1 log CFU/mL of S. aureus was completely inactivated after 14 s IEF treatment. Scanning electron microscopy and transmission electron microscope images showed that IEF caused the destruction of cell membrane and internal substructure, and the damage to intracellular substructure was more severe. Altered membrane integrity or permeability was demonstrated through flow cytometry and confocal laser scanning microscope analysis, and the different damage to cells was quantified by propidium iodide & 5-carboxy fluorescein diacetate single and double staining. In addition, IEF treatment also decreased the membrane potential and esterase activity of S. aureus cells. Putative inactivation mechanism of IEF against S. aureus is a complex process, and its apoptosis is the result of the combination of several factors, which provide a basis for understanding the inactivation mechanism of IEF.

Genetic and compositional analysis of biofilm formed by Staphylococcus aureus isolated from food contact surfaces.

Staphylococcus aureus is an important pathogen that can form biofilms on food contact surfaces (FCS) in the dairy industry, posing a serious food safety, and quality concern. Biofilm is a complex system, influenced by nutritional-related factors that regulate the synthesis of the components of the biofilm matrix. This study determines the prevalence of biofilm-associated genes and evaluates the development under different growth conditions and compositions of biofilms produced by S. aureus. Biofilms were developed in TSB, TSBG, TSBNaCl, and TSBGNaCl on stainless-steel (SS), with enumeration at 24 and 192 h visualized by epifluorescence and scanning electron microscopy (SEM). The composition of biofilms was determined using enzymatic and chemical treatments and confocal laser scanning microscopy (CLSM). A total of 84 S. aureus (SA1-SA84) strains were collected from 293 dairy industry FCS (FCS-stainless steel [n = 183] and FCS-polypropylene [n = 110]) for this study. The isolates harbored the genes sigB (66%), sar (53%), agrD (52%), clfB/clfA (38%), fnbA/fnbB (20%), and bap (9.5%). 99. In particular, the biofilm formed by bap-positive S. aureus onto SS showed a high cell density in all culture media at 192 h in comparison with the biofilms formed at 24 h (p < 0.05). Epifluorescence microscopy and SEM revealed the metabolically active cells and the different stages of biofilm formation. CLSM analysis detected extracellular polymeric of S. aureus biofilms on SS, such as eDNA, proteins, and polysaccharides. Finally, the level of detachment on being treated with DNase I (44.7%) and NaIO 4(42.4%) was greater in the biofilms developed in TSB compared to culture medium supplemented with NaCl at 24 h; however, there was no significant difference when the culture medium was supplemented with glucose. In addition, after treatment with proteinase K, there was a lower level of biomass detachment (17.7%) of the biofilm developed in TSBNaCl (p < 0.05 at 24 h) compared to that in TSB, TSBG, and TSBGNaCl (33.6, 36.9, and 37.8%, respectively). These results represent a deep insight into the composition of S. aureus biofilms present in the dairy industry, which promotes the development of more efficient composition-specific disinfection strategies.

Platelet Membrane Cloaked Nanotubes to Accelerate Thrombolysis by Thrombus Clot-Targeting and Penetration.

Thrombotic diseases have a high rate of mortality and disability, and pose a serious threat to global public health. Currently, most thrombolytic drugs especially protein drugs have a short blood-circulation time, resulting in low thrombolytic efficiency. Therefore, a platelet membrane (Pm) cloaked nanotube (NT-RGD/Pm) biomimetic delivery system with enhanced thrombolytic efficiency is designed. Nanotubes (NT) with an excellent clot-penetration properties are used to load a protein thrombolytic drug urokinase (Uk). Platelet-targeting arginine glycine-aspartic peptide (RGD) is grafted onto the surface of the nanotubes (NT-RGD) prior to cloaking. Multiple particle tracking (MPT) technique and confocal laser scanning microscope (CLSM) analysis are applied and the results show that the nanotubes possess a strong penetration and diffusion capacity in thrombus clots. After the Pm cloaking on NT-RGD/Uk, it shows a thrombus microenvironmental responsive release property and the half-life of Uk is six times longer than that of free Uk. Most importantly, NT-RGD-Uk/Pm exhibits a 60% thrombolytic efficiency in the FeCl3 -induced thrombosis mouse model, and it is able to significantly reduce the bleeding side effects of Uk. This Pm-cloaked nanotube system is an effective and promising platform for the controlled and targeted delivery of drugs for the thrombus treatment.

Green synthesis of carbon quantum dots from plant turmeric holds promise as novel photosensitizer for in vitro photodynamic antimicrobial activity

Research has shown that carbon quantum dots (CQDs) are active as novel carbon nanomaterials in photodynamic therapy due to their excellent photophysical properties. However, previously expensive precursors and time-consuming production processes, as well as complex doping/functionalization forms have limited their economic design and use. In this study, we prepared ST-JHCQDs by a simple and green method using natural plant turmeric as the carbon source, and characterized ST-JHCQDs by physical and optical means. In vitro antibacterial results showed that the antibacterial effects of ST-JHCQDs against E. coli and S. aureus under the irradiation of blue light depended on carbonization degrees, concentration and light duration. Biomolecule leakage and confocal laser scanning microscope analysis showed that ST-JHCQDs were effective in producing reactive oxygen species (ROS) under blue light irradiation, which resulted in disturbance of cell membrane integrity and leakage of intracellular macromolecules in both bacteria. Meanwhile, scanning electron microscopy images showed cell membrane wrinkling and fragmentation, which was consistent with ROS damage, demonstrating the effectiveness of ST-JHCQDs as photosensitizers in vitro photodynamic antimicrobial activity. These experiments show that CQDs prepared from turmeric was a new natural photosensitizer material with great antibacterial potential.

Wheat Glu-A1a encoded 1Ax1 subunit enhances gluten physicochemical properties and molecular structures that confer superior breadmaking quality

Wheat gluten proteins serve as the largest protein molecules in nature and play key roles in breadmaking quality formation. In this study, we used a pair of Glu-A1 allelic variation lines to perform a comprehensive investigation on the effects of Glu-A1a encoded 1Ax1 subunit on gluten physicochemical properties, molecular structures and breadmaking quality. The results showed that the presence of the 1Ax1 subunit significantly increased gluten content, leading to marked improvement of dough rheological properties. Meanwhile, gluten physicochemical properties such as foaming ability and foaming stability, oil/water-holding capacity, emulsifying activity, disulfide bond content, and gluten degradation temperature were significantly improved. A confocal laser scanning microscope analysis revealed that the 1Ax1 subunit drastically enhanced gluten microstructure. Gluten secondary structure analysis by Fourier transform infrared spectroscopy and laser scanning microscope-Raman spectroscopy indicated that 1Ax1 subunit significantly promoted β-turn and β-sheet content and reduced α-helix content. Three-dimensional structure analysis by AlphaFold2 revealed a similar structural feature of 1Ax1 with the superior quality subunit 1Ax2*. Correlation and principal component analyses demonstrated that α-helix and β-sheet content had a significant correlation with dough rheological properties, gluten physicochemical properties and breadmaking quality. Our results showed that 1Ax1 subunit positively affected gluten molecular structure and quality formation.

Abstract 12676: Psychological Stress Stimulates Vascular Inflammatory Responses and Destabilizes Atherosclerotic Plaques as Assessed by High-Speed, High-Resolution Intravital Imaging

Backgrounds: Psychological stress increases leukocyte accumulation within atherosclerotic lesions and exacerbates plaque vulnerability. However, the stress-induced real-time behavior of immune cells in the atheroma has been poorly defined in vivo . Here, we aim to investigate whether stress stimulates the inflammatory leukocyte dynamics in the atherosclerotic plaques and destabilizes the lesions using customized in vivo cell tracking strategies. Methods and Results: We developed a system and motion reconstruction algorithm that can probe and compensate for respiratory and pulsatile movements. Individual leukocytes near the atherosclerotic plaques were imaged in real-time by adapting a custom-built high-speed intravital microscopy system with multiple fluorescence channels. Stress was achieved by immobilization procedures and/or stereotaxic application of stress stimulus onto the brain amygdala. The high spatial and temporal resolution of our real-time cell tracking system allowed clear identification of rhodamine 6G-positive leukocytes in vivo . In the common femoral artery bifurcation of apolipoprotein E knockout mice, white blood cells firmly adhered to the inner layer of the vessel walls while some slowly flowed along the endothelium (Figure). We further demonstrate that the stress increased the rolling and adhesion of inflammatory leukocyte subsets near the atherosclerotic lesions, and enhanced the plaque macrophage activity as assessed by in vivo imaging. Confocal laser scanning microscopy and immunostaining analyses corroborated the in vivo findings that the stress induced the destabilization of the atherosclerotic plaques. Conclusion: Our data show that stress stimulated the dynamics of inflammatory leukocyte subsets in atherosclerotic environments and increased the plaque vulnerability as assessed by the customized high-resolution motion-compensated in vivo imaging strategy.

Encapsulation of rutin in Pickering emulsions stabilized using octenyl succinic anhydride (OSA) modified quinoa, maize, and potato starch nanoparticles

Pickering emulsions (PEs) stabilized with octenyl succinic acid (OSA) modified starch nanoparticles (SNPs) were investigated as a delivery system for rutin (a polyphenol). The nanoscale quinoa (Q, 92 nm), maize (M, 118 nm), and potato (P, 200 nm) starches were prepared by nanoprecipitation and they were OSA-modified as stabilizers. The PEs showed neither coalescence nor Ostwald ripening, as reflected by emulsion index, droplet size, and zeta- potential measurements. Confocal laser scanning microscopic analysis revealed rutin entrapped droplets surrounded by SNPs layer. Fourier-transform infrared spectroscopy, colorimetry, and rheological measurements confirmed the SNPs and rutin interactions, rutin retention, strong network formation, and long-term PE stability. In vitro studies (pH 7.4, 96 h) showed sustained rutin release from the PEs. After 31 days, the encapsulation efficiencies for PEs stabilized with QSNPs, MSNPs, and PSNPs were 99.3, 96.1, and 97.2 %, respectively. The results showed that rutin could be feasibly encapsulated in PEs stabilized with food-grade modified SNPs.

Design of nanostructured coating to prevent biofilm formation on surfaces

Biofilms are surface-attached microbial organizations, where microbial species are enclosed in an exo-polysaccharides matrix. Its removal/prevention from surfaces represents an important challenge and a relevant issue in many fields. In this light, antimicrobial peptides (AMPs) represent good candidates to conventional antibiotics due to their selectivity towards targets (cell membranes), rapid action and low tendency to induce bacterial resistance. In our project we are investigating novel techniques to realise nanostructured coatings on surfaces, in order to prevent bacterial adhesion and biofilm formation. We are focusing our attention on a well-known antimicrobial peptide (Magainin-2). Two bacteria models have been considered: E.coli and P. fluorescens. Our goal is to optimize AMPs grafting on surfaces by means of treatment methodologies based on Oxygen plasma technologies. In this work we present a methodology to investigate anti-biofilm efficiency of the nano-functionalized surfaces, compared with the case of the raw surfaces. Biofilm morphologies can be measured using Confocal Laser Scanning Microscopy and image analysis techniques to quantify its structure by measuring quantitative morphological parameters.

Trans-cinnamaldehyde-Biosurfactant Complex as a Potent Agent against Enterococcus faecalis Biofilms.

Enterococcus faecalis is an opportunistic microbial pathogen frequently associated with diverse infections, including those of the skin and teeth, as well as those from surgical wounds. It forms robust biofilms that are highly tolerant to most antimicrobials and first-line antibiotics. Therefore, investigating alternative strategies to eradicate its biofilms is a critical need. We recently demonstrated that trans-cinnamaldehyde (TC) potently kills E. faecalis biofilm cells and prevents biofilm recovery, and yet, the extreme hydrophobicity of TC hampers clinical translation. Here, we report that a complex of TC with an FDA-approved biosurfactant (acidic sophorolipid/ASL) significantly reduces the bacterial viability and biomass of E. faecalis biofilms, compared to TC alone. A confocal laser-scanning microscopic analysis demonstrated that the TC-ASL treatment significantly decreased the biofilm thickness and volume. In conclusion, our study highlights the anti-biofilm potential of the newly developed TC-ASL.

Potential Application of Luteolin as an Active Antibacterial Composition in the Development of Hand Sanitizer Products

Antibacterial hand sanitizers could play a prominent role in slowing down the spread and infection of hand bacterial pathogens; luteolin (LUT) is potentially useful as an antibacterial component. Therefore, this study elucidated the antibacterial mechanism of LUT against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and developed an antibacterial hand sanitizer. The results showed that LUT had excellent antibacterial activity against both E. coli (minimum inhibitory concentration (MIC) = 312.5 μg/mL, minimal bactericidal concentration (MBC) = 625 μg/mL), and S. aureus (MIC = 312.5 μg/mL, MBC = 625 μg/mL). Furthermore, LUT induced cell dysfunction in E. coli and S. aureus, changed membrane permeability, and promoted the leakage of cellular contents. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) analysis showed that LUT treatment affected cell structure and disrupted cell membrane integrity. The Fourier transform infrared analysis (FTIR) also confirmed that the LUT acted on the cell membranes of both E. coli and S. aureus. Overall, the application of LUT in hand sanitizer had better inhibition effects. Therefore, this study could provide insight into expanding the application of LUT in the hand sanitizer markets.