Confocal Laser Scanning Electron Microscopy Research Articles

From Antagonism to Enhancement: Triton X-100 Surfactant Affects Phenanthrene Interfacial Biodegradation by Mycobacteria through a Shift in Uptake Mechanisms.

Polycyclic aromatic hydrocarbons (PAHs), as persistent environmental pollutants, often reside in nonaqueous-phase liquids (NAPLs). Mycobacterium sp. WY10, boasting highly hydrophobic surfaces, can adsorb to the oil-water interface, stabilizing the Pickering emulsion and directly accessing PAHs for biodegradation. We investigated the impact of Triton X-100 (TX100) on this interfacial uptake of phenanthrene (PHE) by Mycobacteria, using n-tetradecane (TET) and bis-(2-ethylhexyl) phthalate (DEHP) as NAPLs. Interfacial tension, phase behavior, and emulsion stability studies, alongside confocal laser scanning microscopy and electron microscope observations, unveiled the intricate interplay. In surfactant-free systems, Mycobacteria formed stable W/O Pickering emulsions, directly degrading PHE within the NAPLs because of their intimate contact. Introducing low-dose TX100 disrupted this relationship. Preferentially binding to the cells, the surfactant drastically increased the cell hydrophobicity, triggering desorption from the interface and phase separation. Consequently, PAH degradation plummeted due to hindered NAPL access. Higher TX100 concentrations flipped the script, creating surfactant-stabilized O/W emulsions devoid of interfacial cells. Surprisingly, PAH degradation remained efficient. This paradox can be attributed to NAPL emulsification, driven by the surfactant, which enhanced mass transfer and brought the substrate closer to the cells, despite their absence at the interface. This study sheds light on the complex effect of surfactants on Mycobacteria and PAH uptake, revealing an antagonistic effect at low concentrations that ultimately leads to enhanced degradation through emulsification at higher doses. These findings offer valuable insights into optimizing bioremediation strategies in PAH-contaminated environments.

Insight into the inhibitory activity and mechanism of bovine cathelicidin BMAP 27 against Salmonella Typhimurium

This study synthesized an antimicrobial peptide based on the bovine cathelicidin BMAP 27 sequence. It was found to have a broad spectrum of antibacterial activity, with exceptionally high activity against Salmonella. However, the antibacterial mechanism of BMAP 27 against Salmonella remains unclear. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of BMAP 27 against Salmonella enterica serovar Typhimurium were determined to be 2 μM and 4 μM, respectively. After treatment with 2 MIC of BMAP 27, the absorbance of DNA in centrifugal supernatant increased from 0.244 to 1.464, and that of protein rose from 0.174 to 0.774, respectively. BMAP 27 has compromised the cell membrane as observed through field emission scanning electron microscope (FESEM) and transmission electron microscopy (TEM), and confirmed by the propidium iodide (PI) test. The alkaline phosphatase (AKP) enzyme activity in the supernatant of the 2 MIC treatment group was 2.15 times higher than the control group, indicating extracellular membrane damage. BMAP 27 treatment increased intracellular ROS levels as tested by dichlorofluorescein diacetate (DCFH) staining. DNA interaction analysis revealed that BMAP 27 has a binding affinity towards DNA, causing its characteristic bands to disappear and peak intensity at 260 nm to reduce. Molecular docking identified its potential binding mode with DNA. The crystal violet biofilm staining results demonstrated that BMAP 27 inhibited S. Typhimurium biofilm formation by 43.1 % and cleared mature biofilms by 53.62 %. Confocal Laser scanning electron microscopy (CLSM) observed that BMAP 27 could kill bacteria within the biofilm and dislodge bacteria from the surface of glasses. Swimming tests identified that the motor capacity of S. Typhimurium was diminished by BMAP 27. By counting the total bacteria, BMAP 27 was revealed to exert bacteriostatic effects in chilled pork and orange juice, which might provide a basis for its application in the inhibition of Salmonella.

Comparative assessment of nanosized intracanal medicaments on penetration and fracture resistance of root dentin - An in vitro study.

The antimicrobial potential of conventional medicaments is reduced due to their limited permeability. The use of calcium hydroxide (CH) is known to reduce the root strength. Nano intracanal medicaments have the following advantages over conventional such as higher surface area and chemical reactivity, and due to its nanosize, have better penetrability, supporting its possible use as an intracanal medicament. Nano versions of CH, chitosan (CS), CS + CH, curcumin (T), and its conventional forms are used in our study. The aim of this study was to evaluate and compare the depth of penetration of nanosized intracanal medicaments and their effect on fracture resistance of root dentin. Eighty extracted single-rooted teeth were used after decoronation. Canals were enlarged up to size 30 (0.04° taper) using Neoendo instruments (Orikam, India). Teeth were randomly assigned to two broad groups based on the type of intracanal medicament used; Group A: control (conventional) (n = 40) and Group B: nanosized intracanal medicaments (n = 40). Each group was subdivided into four experimental groups (n = 10), Group A1: CH, Group A2: CS, Group A3: curcumin (T), Group A4: CS + CH, Group B1: nano calcium hydroxide (NCH), Group B2: nano CS (NCS), Group B3: nano curcumin (NT), and Group B4: NCS + nano calcium hydroxide (NCS + NCH). All the specimens were stored in a humidor at 37°C for 4 weeks. Out of 10 specimens from each group, five specimens were used for evaluating the depth of penetration using a confocal laser scanning electron microscope, and the remaining five specimens were used for evaluating fracture resistance in a universal testing machine at the end of 4 weeks. Data were analyzed using one-way ANOVA and intergroup comparison using Tukey's post hoc multiple comparison test. The highest fracture resistance was seen with NCS and the maximum depth of penetration with nano calcium hydroxide (NCH). The fracture resistance and depth of penetration at a 4-week interval were higher in nano forms compared to their micro-sized counterparts with all groups. The coronal section presented the highest depth of penetration of intracanal medicaments followed by the middle and least in the apical section of root dentin with all groups.

Time dependent inhibition of Morinda citrifolia essential oils against multi drug resistant bacteria and A549 lung cancer cells

Recent years, multi-drug resistant bacteria (MDRs) and lung cancer cells are threatening to human worldwide due to failure of sufficient drugs. It was suggested that the new sources needed to discover new varieties of drugs, antibiotics and other format to eradicate these infections. As like, the medicinal plant of Morinda citrifolia (M. citrifolia) was used in this study to inhibit both the bacterial infection and cancer cells. First, the LC-MS measurement was evidently reported the available essential oils from medicinal plant of M. citrifolia. It has excellent anti-bacterial properties against P. mirabilis, E. coli, P. aeruginosa and S. aureus with 24, 22, 26 and 18 zone of inhibition respectively by agar well diffusion. Subsequently, the liquid interference assay was exhibited 97 %, 96 %, 94 % and 90 % inhibition at 1000 µg/mL concentration for all the tested pathogens. In addition, M. citrifolia EOs have excellent anti-bacterial activity, and it arrested the bacterial growth completely at 1000 µg/mL concentration for 24 h after compared with other time intervals. Subsequently, the intracellular membranes and more death cells with condensed chromosome like structure were effectively observed by confocal laser scanning electron microscope. Then, invitro cytotoxicity result of M. citrifolia EOs treated A549 lung cancer cells was clearly observed. The cytotoxicity result confirmed that the 500 µg/mL of M. citrifolia EOs against A549 lung cancer cells as fixed as IC50 dose. Hence, current study conclude, M. citrifolia is the excellent medicinal plant to inactivate bacterial growth and cancer cells efficiently.

A pilot study comparing the disinfecting effects of commercialized stable ClO2 solution (free of activation) with conventional H2O2 on dental unit waterlines in the dental practice setting.

Disinfection of dental unit waterlines (DUWLs) plays a key role in control and prevention of nosocomial infection in a dental clinic. The most conventional disinfectant is hydrogen peroxide (H2O2), while chlorine dioxide (ClO2) has been considered however was limited by the "activation" procedures. With the availability of commercialized stable ClO2 solution (free of activation), direct application of ClO2 in the dental practice became possible. This study was designed to compare the disinfecting effects of stable 5 ppm of ClO2 solution with conventional 0.24% of H2O2 on DUWLs in dental practice. Studies of colony-forming units (CFUs), confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM) were employed for evaluation. In CFUs studies, we found that the efficiency of ClO2 was no less than those of H2O2. In the morphological studies, the stronger disinfecting effects of ClO2 was verified by both CLSM and SEM studies for removal and prevention of biofilm. Importantly, ClO2 solution achieved a better disinfecting efficiency not only at the surface of bacterial biofilm, but also, it has penetrating effects, presented disinfecting effects from the surface to the bottom of the biofilm. This pilot study provided evidence regarding the efficiency of stable ClO2 solution on disinfection of DUWLs in the dental practice setting. Application of stable ClO2 solution in dental practice is therefore become possible.

Green synthesized AgNPs, CuONPs, and Ag‐CuO NCs to effective antibiofilm activities and their potential application in aquaculture

The expanded emergence of drug‐resistant microbes remains a concern to the scientific community in developing effective therapeutics. Approach of green chemistry based nanomaterials fabrication is in high demand, due to biocompatibility, non‐toxic, and high surface area that adheres more biomolecules on surface. Green fabricated silver nanoparticles (AgNPs), copper oxide nanoparticles (CuONPs), and silver–copper oxide nanocomposites (Ag‐CuO NCs) were characterized by fluorescence and Raman spectroscopy, field emission scanning electron microscopy (FE‐SEM) with energy‐dispersive X‐ray spectroscopy (EDX), and thermogravimetric (TG)/differential scanning calorimetry (DSC) analysis. Antibiofilm potential of green fabricated AgNPs, CuONPs, and Ag‐CuO NCs was tested against aquatic pathogenic microorganisms such as Aeromonas hydrophila, Pseudomonas aeruginosa, and Staphylococcus aureus. This present study deals with bacterial growth rate, biofilm inhibition, and antibiofilm architecture of AgNPs, CuONPs, and Ag‐CuO NCs under the visualization of confocal laser scanning electron microscopy (CLSM). The FE‐SEM technique was used to observe dense and compact structure (control without NPs treatment) with treatment of NPs that showed reduced amount of cell population in A. hydrophila, P. aeruginosa, and S. aureus. The effective antibiofilm inhibition of Ag‐CuO NCs was achieved by reducing micro‐colonies compared with AgNPs and CuONPs. The AgNPs, CuONPs, and Ag‐CuO NCs demonstrated antibiofilm properties against Gram‐negative and Gram‐positive bacteria, indicating a combinatorial effect of green fabricated NPs in combination with plant metabolites against pathogenic bacteria. As a result, aforementioned nano‐product merits further investigation in order to be used as a potent adjuvant therapy as antagonistic toward various bacterial diseases in aquaculture.

Anti-quorum Sensing and Anti-biofilm Effect of Nocardiopsis synnemataformans RMN 4 (MN061002) Compound 2,6-Di-tert-butyl, 1,4-Benzoquinone Against Biofilm-Producing Bacteria.

In this study, the anti-biofilm compound of 2,6-Di-tert-butyl, 1,4-benzoquinone was purified from Nocardiopsis synnemataformans (N. synnemataformans) RMN 4 (MN061002). To confirm the compound, various spectroscopy analyses were done including ultraviolet (UV) spectrometer, Fourier transform infrared spectroscopy (FTIR), analytical high-performance liquid chromatography (HPLC), preparative HPLC, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), and 2D nuclear magnetic resonance (NMR). Furthermore, the purified compound was shown 94% inhibition against biofilm-producing Proteus mirabilis (P. mirabilis) (MN396686) at 70µg/mL concentrations. Furthermore, the metabolic activity, exopolysaccharide damage, and hydrophobicity degradation results of identified compound exhibited excellent inhibition at 100µg/mL concentration. Furthermore, the confocal laser scanning electron microscope (CLSM) and scanning electron microscope (SEM) results were shown with intracellular damages and architectural changes in bacteria. Consecutively, the in vivo toxicity effect of the compound against Artemia franciscana (A. franciscana) was shown to have a low mortality rate at 100µg/mL. Finally, the molecular docking interaction between the quorum sensing (QS) genes and identified compound clearly suggested that the identified compound 2,6-Di-tert-butyl, 1,4-benzoquinone has anti-quorum sensing and anti-biofilm activities against P. mirabilis (MN396686).

Cladosporium sphaerospermum extract inhibits quorum sensing associated virulence factors of Serratia marcescens

Serratia marcescens is now becoming a propensity for its highly antimicrobial-resistant clinical infections. Currently, it provides a novel strategy to prevent and control microbial infection by regulating S. marcescens quorum sensing (QS). Deep-sea-derived fungi are rich in QS bioactive constituents. In this work, the extracts from Cladosporium sphaerospermum SCSGAF0054 showed potent QS-related virulence factors and biofilm-inhibiting activities against S. marcescens NJ01. The swimming motility and multiple virulence factors such as prodigiosin, exopolysaccharide (EPS), lipase, protease and hemolysin were moderately inhibited by the extracts at varied concentrations. The confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) images revealed that C. sphaerospermum extracts moderately arrested biofilm formation and cell viability. Further, real-time quantitative PCR (RT-qPCR) analysis revealed that expressions of genes associated with virulence factors, flhD, fimA, fimC, bsmA, bsmB, pigA, pigC, and shlA, were significantly down-regulated compared with control. In addition, the extracts combined with imipenem inhibited the QS system of S. marcescens NJ01, disrupted its preformed biofilm, released the intra-biofilm bacteria and killed the bacteria gradually. Therefore, the extracts combined with imipenem can partially restore bacterial drug sensitivity. These results suggest that the extracts from SCSGAF0054 effectively interfere with the QS system to treat S. marcescens infection alone or combining with classical antimicrobial drugs.

Sustainable fluorescent dye-faujasite zeolite systems as tools for cancer bioimaging

The use of fluorescent nanoparticles for biological and medical applications has attracted great attention due to their several advantages compared to conventional fluorescent dyes. However, there is also a need for sustainable and versatile alternatives, alongside the streamlining of complex synthesis or preparations. In this work, we study the potential of the faujasite zeolite structure to encapsulate dye molecules and be applied as a bioimaging agent for cancer cells. The commercial zeolites NaY, USY, and NaX were used to encapsulate Rhodamine B (RhB) or Fluorescein (F) dyes, which present a similar tetracycle core structure. Different characterization techniques confirmed the encapsulation of the dyes and their stability. The photophysical characterization of the encapsulated RhB and F dyes was also elucidated by UV–visible and fluorescence spectroscopy. Biocompatibility was tested in different cancer cell lines and confocal laser scanning electron microscopy (CLSM) was used to track the fluorescent dye-containing zeolites. Remarkably, the dye-containing zeolite RhB@USY showed interesting multiluminescent labeling properties that make it a suitable agent for bioimaging applications. The economic feasibility of the developed fluorescent dye-zeolite probes was also examined through a detailed cost analysis, which further highlights their commercial potential.

Sunlight-driven intimately coupled photocatalysis and biodegradation (SDICPB): A sustainable approach for enhanced detoxification of triclosan

Triclosan is considered as recalcitrant contaminant difficult to degrade from the contaminated wastewater. Thus, promising, and sustainable treatment method is necessary to remove triclosan from the wastewater. Intimately coupled photocatalysis and biodegradation (ICPB) is an emerging, low-cost, efficient, and eco-friendly method for the removal of recalcitrant pollutants. In this study BiOI photocatalyst coated bacterial biofilm developed at carbon felt for efficient degradation and mineralization of triclosan was studied. Based on the characterization of BiOI prepared using methanol had lower band gap 1.85 eV which favors lower recombination of electron-hole pair and higher charge separation which ascribed to enhanced photocatalytic activity. ICPB exhibits 89% of triclosan degradation under direct sunlight exposure. The results showed that production of reactive oxygen species hydroxyl radical and superoxide radical anion played crucial role in the degradation of triclosan into biodegradable metabolites further the bacterial communities mineralized the biodegradable metabolites into water and carbon dioxide. The confocal laser scanning electron microscope results emphasized that interior of the biocarrier shows a large number of live bacterial cells existing in the photocatalyst-coated carrier, where the little toxic effect on bacterial biofilm occurred on the exterior of the carrier. The extracellular polymeric substances characterization result remarkable confirms that which could act as sacrificial agent of photoholes further helped by preventing the toxicity to the bacterial biofilm from the reactive oxygen species and triclosan. Hence, this promising approach can be a possible alternative method for the wastewater treatment polluted with triclosan.

Green synthesis of silver nanoparticles using Eucommia ulmoides leaf extract for inhibiting stem end bacteria in cut tree peony flowers.

Tree peony ( Paeonia suffruticosa Andr.) is a popular cut flower among ornamental plants. However, its short vase life severely hinders the production and application of cut tree peony flowers. To extend the postharvest longevity and improve the horticultural value, silver nanoparticles (Ag-NPs) was applied for reducing bacterial proliferation and xylem blockage in cut tree peony flowers in vitro and in vivo. Ag-NPs was synthesized with the leaf extract of Eucommia ulmoides and characterized. The Ag-NPs aqueous solution showed inhibitory activity against bacterial populations isolated from stem ends of cut tree peony 'Luoyang Hong' in vitro. The minimum inhibitory concentration (MIC) was 10 mg L-1. Compared with the control, pretreatments with Ag-NPs aqueous solution at 5 and 10 mg L-1 for 24h increased flower diameter, relative fresh weight (RFW), and water balance of tree peony 'Luoyang Hong' flowers. Additionally, malondialdehyde (MDA) and H2O2 content in pretreated petals were lower than the control during the vase life. The activities of superoxide dismutase (SOD) and catalase (CAT) in pretreated petals were lower than that of the control at the early vase stage and higher at the late vase life. Furthermore, pretreatments with Ag-NPs aqueous solution at 10 mg L-1 for 24h could reduce bacterial proliferation in the xylem vessels on the stem ends by confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Overall, pretreatments with green synthesized Ag-NPs aqueous solution effectively reduced bacteria-induced xylem blockage of cut tree peony, resulting in improved water uptake, extended vase life, and enhanced postharvest quality. Therefore, this technique can be used as a promising postharvest technology in the cut flower industry.

The role of β-subunit in emulsifying performance of β-conglycinin

The most crucial protein that affects the ability of soybean protein to emulsify is β-conglycinin. The subunit composition of β-conglycinin is one of the important factors affecting its emulsification performance. In order to investigate the effect of β-subunit of β-conglycinin on its emulsifying property, the separated and purified β-subunit was added to β-conglycinin in different proportions, and the properties of the β-conglycinin before and after emulsification were measured. The β-subunit appeared to improve the stability of the β-conglycinin in the aqueous dispersion as evidenced by the average particle size significantly decreasing, the surface hydrophobicity significantly increasing, and the Emulsification activity index and Emulsion stability index further improving. The secondary structure of β-conglycinin was altered by the addition of β-subunit, which caused more conversion of β-sheet and β-turn to α-helix, increasing the flexibility of protein molecules. Additionally, the particle size distribution, ζ-potential and creaming index of the stable emulsion formed by β-conglycinin were also measured. The results showed that the increase of β-subunit content improved the stability of the emulsion to some extent. The stability of emulsion mainly relies on their internal structure to maintain. It could be concluded from rheological analysis that the viscosity of emulsion increases due to the presence of β-subunits and the existence of weak gel structure inside them, which is corroborated by the results of confocal laser scanning electron microscopy. The elucidation of the influence of subunits on the emulsification characteristics of soyabean protein is beneficial to the development of soybean protein industry.

OBSERVAÇÃO E ANÁLISE DE IMAGEM BASEADA EM SENSORES VISUAIS DAS LESÕES MUSCULARES EM ATLETAS

ABSTRACT Introduction: Athletes and fitness enthusiasts often suffer muscle injuries during their training. These injuries can worsen when not treated properly, generating an accumulation of severe tissue damage, preventing optimal physical performance, and impacting low immunity. Despite a large number of researches on muscle injuries, its vast majority is limited to the pathological perspective, and there are few studies related to the specific impact of the body sport muscle injury index. Objective: Analyze the body-specific indicators of sports injury to prevent further damage to its practitioners. Methods: Laser scanning confocal electron microscopy is the main observation tool in studying muscle injury in athletes. In further research, an experimental animal model was established. The test samples were 40 male rats over 12 weeks old, randomly divided into four groups, treadmill exercise, swimming at 8% of the weight, and other sports training items. Results: Through the comparative experiment of three indicators, it is found that muscle damage has a widespread impact on the whole body, particularly on blood serum indicators. The period of one hour after injury is considered to have the most impact. However, the symptoms subside after 24 hours. Conclusion: As the main observation tool in this experiment, the microscope showed its good prospect of application in the field of biomedicine. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

Photodynamic toluidine blue-gold nanoconjugates as a novel therapeutic for Staphylococcal biofilms.

Staphylococci are among the most frequent bacteria known to cause biofilm-related infections. Pathogenic biofilms represent a global healthcare challenge due to their high tolerance to antimicrobials. In this study, water soluble polyethylene glycol (PEG)-coated gold nanospheres (28 ppm) and nanostars (15 ppm) with electrostatically adsorbed photosensitizer (PS) Toluidine Blue O (TBO) ∼4 μM were successfully synthesized and characterized as PEG-GNPs@TBO and PEG-GNSs@TBO. Both nanoconjugates and the TBO 4 μM solution showed remarkable, if similar, antimicrobial photodynamic inactivation (aPDI) effects at 638 nm, inhibiting the formation of biofilms by two Staphylococcal strains: a clinical methicillin-resistant Staphylococcus aureus (MRSA) isolate and Staphylococcus epidermidis (S. epidermidis) RP62A. Alternatively in biofilm eradication treatments, the aPDI effects of PEG-GNSs@TBO were more effective and yielded a 75% and 50% reduction in viable count of MRSA and S. epidermidis RP62A preformed biofilms, respectively and when compared with untreated samples. This reduction in viable count was even greater than that obtained through aPDI treatment using a 40 μM TBO solution. Confocal laser microscopy (CLSM) and scanning electron microscope (SEM) images of PEG-GNSs@TBO's aPDI treatments revealed significant changes in the integrity and morphology of biofilms, with fewer colony masses. The generation of reactive oxygen species (ROS) upon PEG-GNSs@TBO's aPDI treatment was detected by CLSM using a specific ROS fluorescent probe, demonstrating bright fluorescence red spots across the surfaces of the treated biofilms. Our findings shine a light on the potential synergism between gold nanoparticles (AuNPs) and photosensitizers in developing novel nanoplatforms to target Staphylococcal biofilm related infections.

Effects of Luteolin on Biofilm of Trueperella pyogenes and Its Therapeutic Effect on Rat Endometritis.

Trueperella pyogenes is an opportunistic pathogen that causes suppurative infections in animals. The development of new anti-biofilm drugs will improve the current treatment status for controlling T. pyogenes infections in the animal husbandry industry. Luteolin is a naturally derived flavonoid compound with antibacterial properties. In this study, the effects and the mechanism of luteolin on T. pyogenes biofilm were analyzed and explored. The MBIC and MBEC of luteolin on T. pyogenes were 156 μg/mL and 312 μg/mL, respectively. The anti-biofilm effects of luteolin were also observed by a confocal laser microscope and scanning electron microscope. The results indicated that 312 μg/mL of luteolin could disperse large pieces of biofilm into small clusters after 8 h of treatment. According to the real-time quantitative PCR detection results, luteolin could significantly inhibit the relative expression of the biofilm-associated genes luxS, plo, rbsB and lsrB. In addition, the in vivo anti-biofilm activity of luteolin against T. pyogenes was studied using a rat endometritis model established by glacial acetic acid stimulation and T. pyogenes intrauterine infusion. Our study showed that luteolin could significantly reduce the symptoms of rat endometritis. These data may provide new opinions on the clinical treatment of luteolin and other flavonoid compounds on T. pyogenes biofilm-associated infections.

Adherent Bacteria and Parasiticidal Secretion Products of Human Cervicovaginal Microbiota-Associated Lactobacillus gasseri Confer Non-Identical Cell Protection against Trichomonas vaginalis-Induced Cell Detachment.

Trichomonas vaginalis, a protozoan parasite specific to the human genital tract, is one of the most common sexually transmitted pathogens. Its pathogenicity is strongly associated with its expression of a broad array of proteases triggering cytotoxic effects in host epithelial cells. Vaginal microbiota-associated Lactobacillus, including those of L. gasseri in particular, can counteract T. vaginalis pathogenesis, but the mechanisms involved have yet to be clarified. T. vaginalis strain G3 (Tv G3) cytotoxicity was assessed by examining cell morphology, cell detachment, and fluorescent labeling of the F-actin cytoskeleton and immunolabeling of vinculin-position focal adhesions (FAs) by confocal laser scanning electron microscopy on confluent cervicovaginal epithelial HeLa cell monolayers. The inhibitory effects of bacterial cells and secreted products of L. gasseri ATCC 9857 and KS 120.1 on the Tv G3 viability and parasite deleterious effects on HeLa cells were investigated. Pre-adhering L. gasseri cells delayed but did not inhibit Tv G3-induced cell detachment, F-actin cytoskeleton disorganization and the disappearance of vinculin-positive focal FAs. L. gasseri KS 120.1 secretion products had a rapid parasiticide activity by killing time- and concentration-dependent Tv G3 parasites after direct contact. By killing Tv G3 parasites already associated with the epithelial cells, secretion products have abolished parasite-induced cell detachment. Our findings suggest that vagina microbiota-associated L. gasseri creates a physical barrier and exerts pharmacological-type mechanisms to counteract the deleterious cytotoxic effects of T. vaginalis.

Unraveling the mechanism of sulfur nutrition in pigeonpea inoculated with sulfur-oxidizing bacteria.

An investigation was carried out to understand the mechanism(s) involved in the uptake of sulfur (S) as sulfate in pigeonpea following single inoculation of two sulfur-oxidizing bacteria (SOB), Stenotrophomonas maltophilia and Stenotrophomonas pavanii in the treatments amended with either elemental sulfur (S0) or sulfate (S6). Colonization potential and biofilm formation were analyzed through confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Furthermore, the effect of seed inoculation on root architecture, expression of genes involved in sulfur oxidation (sox) in bacterial inoculants, and genes involved in sulfate transport in pigeonpea (PpSULTR) were analyzed to correlate with the higher uptake of S in roots and shoots of pigeonpea. Both the SOB exhibited a good colonization potential and biofilm formation on the roots of pigeonpea. Among the 11 sox genes targeted in rhizosphere of pigeonpea, expression was achieved for seven genes, which showed 2-fold increase in treatments inoculated with S. maltophilia and amended with either S6 or S0. The inoculation of S. maltophilia and amendment of S0 led to increased expression of PpSULTR genes by several folds in roots. The inoculation of SOB had a significant influence on non-enzymatic (osmolytes like proline) and enzymatic (PAL, peroxidase, superoxide dismutase, and catalase) levels. The results revealed a significant increase in sulfur uptake in roots and shoots in treatment inoculated with S. maltophilia and amended with S6. The investigation showed that the SOB-mediated over-expression of PpSULTR genes in roots of pigeonpea and sox genes in the rhizosphere were acting synergistically in facilitating higher uptake and translocation of S in roots and shoots of pigeonpea plants.

Comparison of extraction methods for extracellular polymeric substances (EPS) and dynamic characterization of EPS from sessile microorganisms during pyrite bioleaching

Extracellular polymeric substances (EPS) have many beneficial functions in the bioleaching process. A mixed culture of moderate thermophilic microorganisms was used to bioleach pyrite concentrate in a stirred tank reactor at a pulp density of 6%. Three physical methods (heating, ultrasound and vibration with glass beads) and three chemical methods (NaOH, H2SO4 and formaldehyde) were assessed for the extraction of EPS from sessile microorganisms. The parameter conditions for each method were optimised to obtain the optimal settings. The EPS content of each extract varied depending on the extraction method used. Cell lysis can be minimised by optimizing the extraction conditions. Results indicated that heating method outperformed other methods with low cell lysis and yielded 355.56 ± 116.56 ug/108 cells of protein and 16.72 ± 1.45 ug/108 cells of polysaccharides. Thereafter, this method was applied to extract EPS from pyrite surface during the bioleaching process. During pyrite bioleaching, extracellular proteins were the major component of EPS extracted from sessile strains. Confocal laser scanning microscope and scanning electron microscope showed that pyrite grains were wrapped by many extracellular proteins. Therefore, this research can provide a reference for the extraction of EPS from moderate thermophilic bioleaching systems by analysing the potential impact of extraction methods.

Anti-biofilm studies of synthetic imidazolium salts on dental biofilm in vitro

ABSTRACT Objective Biofilm formation under cariogenic conditions contributes to dental caries development, in which Streptococcus mutans (S. mutans) is regarded as the major cariogenic bacteria. Here, we synthesized a series of imidazolium salts. Their properties of antimicrobial and anti-biofilm were investigated. Methods The microdilution method crystal violet staining, and cell counting Kit-8 assay were used to screen imidazolium salts. Then, the bacterial composition in multi-species biofilm composed of S. mutans, Actinomyces naeslundii, and Streptococcus gordonii was quantified by quantitative PCR. The exopolysaccharide and morphology of the structure of multi-species biofilm were further observed by confocal laser scanning microscopy and scanning electron microscope, respectively. Results Imidazolium salts exhibited highly antimicrobial activity against oral pathogens, especially for S. mutans . Compounds with ortho-diisopropyl and para-methoxyl on N-moieties as well as bearing ancenaphthyl skeleton (C5) showed the lowest cytotoxicity and most efficient anti-biofilm activity. C5 inhibited approximately 50% of multi-species biofilm at 0.98 μg/mL. Notably, C5 resulted in 98.97% live S. mutans and 77.65% A. naeslundii decreased. Furthermore, the exopolysaccharide was reduced by 88%, along with a sparse and scattered microstructure. Conclusion The imidazolium salts present low cytotoxicity and remarkable antimicrobial activity against S. mutans in multi-species biofilm, suggesting that they may have a great potential in anti-biofilm clinical applications.

Bacillus subtilis BR4 derived stigmatellin Y interferes Pqs-PqsR mediated quorum sensing system of Pseudomonas aeruginosa.

Cell-to-cell communication is essentially required in bacteria for the production of multiple virulence factors and successful colonization in the host. Targeting the virulence factors production without hampering the growth of the pathogens is a potential strategy to control pathogenesis. To accomplish this, a total of 43 mangrove isolates were screened for quorum quenching (QQ) activity against Pseudomonas aeruginosa (PA), in which eight bacteria have shown antibiofilm activity without hampering the growth of the PA. Prominent QQ activity was observed in Bacillus subtilis BR4. Previously, we found that BR4 produces stigmatellin Y, a structural analogue of PQS signal of PA, which could competitively bind with PqsR receptor and inhibits the quorum sensing (QS) system of PA. Further, stigmatellin Y containing ethyl acetate extract (S-EAE) (100 µg ml-1 ) of BR4 significantly inhibits (p < 0.001) the biofilm formation of PA. Confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) analysis also fortified the QQ activity of BR4. Furthermore, S-EAE of BR4 (500 µg ml-1 ) has significantly reduced the production of virulence factors, including protease, elastase, pyocyanin and extracellular polysaccharides substances. Furthermore, liquid chromatography-mass spectrometry (LC-MS)/MS analysis affirms that BR4 intercepts the PQS-mediated QS system by reducing the synthesis of as many PQS signals, including precursor molecule (243.162313 Da) of PQS signal. Thus, S-EAE of B. subtilis BR4 could be used as a promising therapeutic agent to combat QS system-mediated pathogenesis of PA. Further therapeutic potentials of stigmatellin Y to be evaluated in clinical studies for the treatment of multidrug resistant PA.