Leakage Of Potassium Ions Research Articles

Antimicrobial Activity and Mechanisms of Punicalagin against Vibrio parahaemolyticus.

This study sought to explore the antimicrobial activity of punicalagin against V. parahaemolyticus and its potential modes of action. V. parahaemolyticus ATCC 17802 and RIMD 2210633Sm were exposed to punicalagin, and the energy production, membrane potential, and envelope permeability, as well as the interaction with cell biomolecules, were measured using a variety of fluorescent probes combined with electrophoresis and Raman spectroscopy. Punicalagin treatment disrupted the envelope integrity and induced a decrease in intracellular ATP and pH. The uptake of 1-N-phenyl-naphtylamine (NPN) demonstrated that punicalagin weakened the outer membrane. Punicalagin damaged the cytoplasmic membrane, as indicated by the membrane depolarization and the leakage of intracellular potassium ions, proteins, and nucleic acids. Electronic microscopy observation visualized the cell damage caused by punicalagin. Further, gel electrophoresis coupled with the Raman spectrum assay revealed that punicalagin affected the protein expression of V. parahaemolyticus, and there was no effect on the integrity of genomic DNA. Therefore, the cell envelope and proteins of V. parahaemolyticus were the assailable targets of punicalagin treatment. These findings suggested that punicalagin may be promising as a natural bacteriostatic agent to control the growth of V. parahaemolyticus.

In vitro Assessment of Antimicrobial potential of Siddha Polyherbal Formulation Tulasi oil against RTI pathogens

In Siddha medication, along with herbal formulations, some mineral formulations are used clinically to prevent or cure infectious diseases. The therapeutic values of some siddha formulations have been well documented earlier, but a huge number of them remain unexplored in terms of safety and efficacy. The aim of this study was to screen the antimicrobial potential of Siddha polyherbal formulation Tulasi oil (SPHTO) against respiratory tract infection (RTI) causing pathogens. The antimicrobial activity of SPHTO was examined by using the methods of agar well diffusion, Minimum inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), Minimum Fungicidal Concentration (MFC) and Microbicidal action. Ciprofloxacin (bacteria) and Amphotericin B (fungi) were used as the positive control for this study. The potassium leakage was determined using a flame emission and atomic absorption spectroscopy. All tested concentrations gave notable antimicrobial activity against all tested pathogens. The 100mg/ml concentration showed the highest activity against E.coli followed by 200mg/ml against K. pnuemonia and C.neoformans when compared to 50 and 25mg/ml. For MIC, the highest activity was registered at 150mg/ml concentration against E. coli, while MBC/MFC was 200mg/ml concentration has shown the better activity against K. pneumonia and C. neoformans. Tulasi oil induced leakage of potassium ions from cells of E.coli and K.pneumonia suspended in glycil-glycine buffer solution and the leakage started within 10 minutes of contact of drugs with target cells. From this study, it may support the clinical claims of Tulasi oil to have potent antibacterial and antifungal activities and offer profound therapeutic benefits against respiratory tract infection.

Synergistic Antibacterial Efficacy of Melittin in Combination with Oxacillin against Methicillin-Resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) often cause infections with high mortality rates. Antimicrobial peptides are a source of molecules for developing antimicrobials; one such peptide is melittin, a fraction from the venom of the Apis mellifera bee. This study aimed to evaluate the antibacterial and antibiofilm activities of melittin and its association with oxacillin (mel+oxa) against MRSA isolates, and to investigate the mechanisms of action of the treatments on MRSA. Minimum inhibitory concentrations (MICs) were determined, and synergistic effects of melittin with oxacillin and cephalothin were assessed. Antibiofilm and cytotoxic activities, as well as their impact on the cell membrane, were evaluated for melittin, oxacillin, and mel+oxa. Proteomics evaluated the effects of the treatments on MRSA. Melittin mean MICs for MRSA was 4.7 μg/mL and 12 μg/mL for oxacillin. Mel+oxa exhibited synergistic effects, reducing biofilm formation, and causing leakage of proteins, nucleic acids, potassium, and phosphate ions, indicating action on cell membrane. Melittin and mel+oxa, at MIC values, did not induce hemolysis and apoptosis in HaCaT cells. The treatments resulted in differential expression of proteins associated with protein synthesis and energy metabolism. Mel+oxa demonstrated antibacterial activity against MRSA, suggesting a potential as a candidate for the development of new antibacterial agents against MRSA.

Green synthesis and characterization of an orally bioactive artemisinin-zinc nanoparticle with enhanced bactericidal activity

The occurrence of multidrug-resistant bacteria necessitates the development of new antibacterial agents. This study synthesized artemisinin-zinc nanoparticles (AZ NPs) using a simple green method and investigated their physicochemical properties, antibacterial activity, and oral biological activity. A spherical shape morphology of AZ NPs was observed by scanning and transmission electron microscopy, with a particle size of 73 ± 2.604 nm. Energy dispersive spectrometry analysis showed that the AZ NPs consisted mainly of Zn, C, N, and O elements. According to differential scanning calorimeter analysis, the AZ NPs were stable up to 450 °C. Fourier-transform infrared spectroscopy revealed that artemisinin successfully bound to zinc acetate. The AZ NPs showed antibacterial activity against Salmonella and Escherichia coli, with a minimum inhibitory concentration of 0.056 mg/mL for both and minimum bactericidal concentrations of 0.21 and 0.11 mg/mL, respectively. The mechanisms by which AZ NPs mediate membrane damage were revealed by the downregulation of gene expression, and potassium ion and protein leakage. In vivo safety trials of these drugs revealed low toxicity. After AZ NPs were administered to infected mice, the intestinal bacteria decreased significantly, liver and kidney function were restored, histopathological damage to the liver and spleen were reduced, and the expression of inflammatory cytokines decreased. Therefore, AZ NPs have the potential as an oral antibacterial agent and can be used in antibiotic development and in the pharmaceutical industry.

Mechanism of Action and Efficiency of Ag3PO4-Based Photocatalysts for the Control of Hazardous Gram-Positive Pathogens.

Silver phosphate and its composites have been attracting extensive interest as photocatalysts potentially effective against pathogenic microorganisms. The purpose of the present study was to investigate the mechanism of bactericidal action on cells of opportunistic pathogens. The Ag3PO4/P25 (AGP/P25) and Ag3PO4/HA (HA/AGP) powders were prepared via a co-precipitation method. Thereafter, their antimicrobial properties against Enterococcus faecalis, Staphylococcus epidermidis, and Staphylococcus aureus (clinical and reference strains) were analyzed in the dark and after exposure to visible light (VIS). The mechanism leading to cell death was investigated by the leakage of metabolites and potassium ions, oxidative stress, and ROS production. Morphological changes of the bacterial cells were visualized by transmission electron microscopy (TEM) and scanning transmission electron microscopy with energy-dispersive X-ray spectroscopy (SEM EDS) analysis. It has been shown that Ag3PO4-based composites are highly effective agents that can eradicate 100% of bacterial populations during the 60 min photocatalytic inactivation. Their action is mainly due to the production of hydroxyl radicals and photogenerated holes which lead to oxidative stress in cells. The strong affinity to the bacterial cell wall, as well as the well-known biocidal properties of silver itself, increase undoubtedly the antimicrobial potential of the Ag3PO4-based composites.

A novel approach for water disinfection by enhanced photoanode oxidation using in-situ generated hydrogen peroxide

To improve disinfection efficiency in distributed water treatment systems using clean energy, a novel photoelectrochemical process was adopted. This process couples photoanodic oxidation with in-situ generated H2O2 (PEC/E-H2O2) and was used for the inactivation of Escherichia coli (E. coli) under simulated solar conditions. In a low-concentration 5 mM Na2SO4 electrolyte, the PEC/E-H2O2 process consumed only 2.14 KJ/L-log of energy and eliminated 106 CFU/mL of E. coli within 50 min. The 0.61 mM H2O2 in-situ generated enhanced the oxidation of the photoelectrochemical system due to the formation of •OH. Compared with traditional electrochemically generated H2O2 (E-H2O2) and photoelectrochemical (PEC) processes, the specific rate constant of E. coli inactivated by the PEC/E-H2O2 process increased by 2.19 and 2.11 times, respectively. The mechanism and the contribution of active species to the process were investigated through a series of competition experiments, fluorescence probes, and EPR measurements. It was verified that the generated radicals, especially h+, •OH, and •O2−, played a significant role in bacterial inactivation. Additionally, the electron transfer mechanism of the PEC/E-H2O2 system and the possible inactivation mechanism of E. coli were proposed. SEM comparisons, potassium ion leakage, and the β-GAL activity decreasing of targeted cells indicated the destruction of the inner and outer cellular membranes, thus causing the inactivation of E. coli. In summary, the study provides a promising alternative disinfection process for drinking water treatment.

Pterostilbene, a Natural Methoxylated Analog of Resveratrol, Exhibits Antifungal Activity Induced by Reactive Oxygen Species Production and Plasma Membrane Injury

Pterostilbene has been shown to exhibit antifungal activities. However, the detailed action mechanism is unknown. Here, we analyzed the antifungal mechanism of pterostilbene against Saccharomyces cerevisiae. The minimum growth inhibitory and fungicidal concentrations of pterostilbene were 120 and 240 µM in a YPD medium, respectively. Although pterostilbene produced reactive oxygen species (ROS), resveratrol did not. This effect was maximized at 120 µM. Furthermore, α-tocopherol acetate inhibited ROS production and reversed pterostilbene-induced growth inhibition. At 240 µM, pterostilbene showed fungicidal effects accompanied by the leakage of intracellular potassium ions, suggesting the involvement of membrane injury in addition to oxidative stress in fungicidal action. Nevertheless, the antioxidants gradually reversed the decrease in cell viability caused by pterostilbene, suggesting that ROS production mainly contributed to the lethal effect. As Pterocarpus marsupium extract, mainly containing pterostilbene, is used as a traditional medicine, pterostilbene has great potential for development as a preservative with fewer adverse effects.

A semi purified hydroalcoholic fraction from Caesalpinia bonduc seeds causes ergosterol biosynthesis inhibition in Candida albicans resulting in cell membrane damage.

Candida species are currently developing resistance to prevailing commercially available drugs, which raises an instantaneous need to discover novel antifungals. To cope with this shocking situation, phytochemicals are the richest, safest, and most potent source of excellent antimicrobials with broad-spectrum activity. The aim of the current study is to explore the anticandidal potential of the various fractions purified from the hydroalcoholic extract of C. bonduc seed. Out of five fractions purified from the hydroalcoholic extract, fraction 3 (Fr. 3) recorded the best activity against C. albicans (8μg/mL) and thus this species was chosen for further mechanism of action studies. The phytochemical examination reveals that Fr. 3 was found to contain steroids and triterpenoids. This was further supported by LC-QTOF-MS and GCMS analyses. Our findings show that Fr. 3 targets the ergosterol biosynthesis pathway in C. albicans by inhibiting the lanosterol 14-α demethylase enzyme and downregulating expression of its related gene ERG11. Molecular docking outcomes disclosed favorable structural dynamics of the compounds, implying that the compounds present in Fr. 3 would be able to successfully bind to the lanosterol 14-α demethylase, as evidenced by the docked compounds' strong interaction with the target enzyme's amino acid residues. Considering virulence factors, the Fr. 3 recorded significant antibiofilm activity as well as germ-tube reduction potential. Furthermore, Fr. 3 enhances the production of intracellular reactive oxygen species (ROS). This suggests that the antifungal activity of Fr. 3 was associated with membrane damage and the induction of ROS production, resulting in cell death. Fluorescence microscopic analysis of PI stained Candida further showed changes in the plasma membrane permeability, which causes severe loss of intracellular material and osmotic balance. This was demonstrated by the potassium ion leakage and release of genetic materials. Finally, the erythrocyte lysis assay confirmed the low cytotoxicity of Fr. 3. Both in silico and in vitro results suggest that Fr. 3 has the potential to propel forward novel antifungal drug discovery programmes.

Cell leakage mechanism and time kill studies on Staphylococcus aureus after exposure to ethanol leaf extract of Muntingia calabura L

Purpose: To determine the effect of the ethanol leaf extract of M. calabura (EEMC) on cell leakage and time-kill against S. aureus. 
 Methods: The leaves were macerated with 96 % ethanol (1:8; w/v) for 27 h to produce EEMC. Chemical compounds of EEMC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Various concentrations of EEMC (12.5; 25; 50; 100 mg/mL) were tested to determine the Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against S. aureus. Furthermore, EEMC was tested for its effect on cell leakage, changes in extracellular electrical conductivity, and time-kill against S. aureus. UV-spectrophotometer was used to test for leakages of nucleic acid, protein, DNA, and RNA, while atomic absorption spectrophotometer was used to evaluate leakage of potassium ion (K+).
 Results: The MIC and MBC of EEMC against S. aureus were 10 % w/v (100 mg/mL). The highest cell leakage occurred in S. aureus exposed to 2x MIC, with leakages of protein, DNA, RNA, and K+ reaching 137.79 ± 58.99, 2298 ± 263.26, 1839 ± 210.61 and 770.86 ± 40.11 μg/mL respectively. The EEMC (1x MBC and 1.5x MBC) killed S. aureus in 24 h. Analysis of LC-MS/MS of EEMC showed that flavonoids (48.33 %) followed by anthraquinones (16.10 %) were the major classes of compounds present in the extract.
 Conclusion: The ethanol leaf extract of M. calabura kills S. aureus by inducing cell leakage possibly due to flavonoids and anthraquinones contained in it. The extract should be further isolated and its active principles with potent antibacterial properties developed for therapeutic applications.

Bilirubin- and Blue-Green Light-Induced Damage of Human Erythrocytes

Phototherapy is widely used for neonatal hyperbilirubinemia treatment; however, the most important but not completely recognized issue is the determination of the optimal spectral range of light that provides an effective reduction of bilirubin level in the blood of a newborn with minimal adverse side effects. This study demonstrates that the exposure of radiation from LED sources of blue (emission wavelength λ max = 463 nm, FWHM 20 nm) and green (λ max = 517 nm, FWHM 38 nm) spectral regions to the erythrocyte suspension with the same energy dose (D = 14.4 J/cm 2 ) leads to approximately equally effective hemolysis of erythrocytes. It is concluded that the damage of erythrocyte membranes is caused by the excitation of endogenous porphyrin and flavin photosensitizers contained in the cells in low concentrations, but characterized by high efficiency of singlet oxygen generation. When bilirubin is added to the erythrocyte suspension, a spontaneous (dark) erythrocyte hemolysis intensifies, but the light-induced hemolysis slows down (compared to cells containing no bilirubin). Under the same conditions, the leakage of potassium ions from light-exposed erythrocytes is accelerating, with the light of green spectral region has a more pronounced effect, despite more intense absorption of blue light by bilirubin localized in cells.

Magnetically recoverable Fe3O4/MoS2/BiOI microspheres for visible light water disinfection: Molecular mechanism and transcriptomic insights

Photocatalysis driven by green energy is ideal for water purification. To gain a deeper understanding of the underlying mechanism behind the photocatalytic inactivation of bacteria, a novel visible-light-driven Fe3O4/MoS2/BiOI (FMB) photocatalyst was synthesized, and E. coli disinfection was demonstrated. Complete inactivation of E. coli was achieved in 100 min by FMB exposed to 30 mW/cm2 of > 400 nm light. FMB can also effectively reduce the total number of bacteria and heterotrophic bacteria in actual source water. The photochemical experiments revealed that h+, e-, H2O2, 1O2 and •O2– were responsible for inactivation reactions. The semipermeable membrane experiments provided further evidence that contact between the photocatalyst and the bacteria was necessary to achieve inactivation. During the disinfection process, the zeta potential of the cells first decreased and then increased, while the particle size first increased and then decreased, indicating the rupture of the cells. Scanning electron microscopy, potassium ion leakage, and changes in cell surface hydrophobicity and hydrophilicity all confirmed the destruction of the E. coli cell membrane at the molecular level. The β-GAL activity of E. coli decreased, and the activities of superoxide dismutase (SOD) and catalase (CAT) increased initially, but subsequently decreased, further demonstrating disruption of the cell membrane and the leakage of cell contents. Transcriptomics was employed to understand gene expression and confirm bacterial membrane damage followed by oxidative stress response. This work provides a demonstration of the FMB inactivation by visible light and a mechanistic understanding of inactivation of E. coli.

Facile preparation and antibacterial activity of zinc oxide nanobullets

Bacterial infections and emergence of many multidrug resistant bacterial pathogens are serious threats to human health. Nanostructured metal oxides are now used as antibacterial additives in various products of everyday usage, but their mode of antibacterial action still remains unclear. ZnO nanobullets were synthesized using a surfactant-free and template-free simple chemical route and well characterized by using FESEM, TEM, HRTEM, XRD, Raman and UV-vis spectroscopy. FESEM and TEM revealed bullet-shaped ZnO nanostructures with average size 50 nm. XRD analysis confirmed hexagonal wurtzite structure of ZnO nanobullets with crystallite size of 24 nm. We explored the antibacterial effects of ZnO nanobullets on gram-negative (Escherichia coli) and gram-positive (Bacillus oceanisediminis), and investigated the underlying mechanism using morphological analysis by SEM, ROS scavenging by histidine, thiobarbituric acid reactive species (TBARS) assay, analysis of potassium ion leakage and Bradford assay. Our results demonstrated that ZnO nanobullets efficiently inhibited the growth of both Escherichia coli and Bacillus oceanisediminis bacteria. Furthermore, various biochemical and molecular assays have provided evidence that the strong antibacterial action of ZnO nanobullets can be attributed to ROS induced oxidative stress resulting in enhanced lipid peroxidation and cell membrane damage, which subsequently caused protein leakage. In summary, our results show that ZnO nanobullets exhibit a strong antibacterial activity against both Escherichia coli and Bacillus oceanisediminis under ambient lighting, which is exciting for practical applications.

Juglone Inhibits Listeria monocytogenes ATCC 19115 by Targeting Cell Membrane and Protein.

Foodborne disease caused by Listeria monocytogenes is a major global food safety problem. A potential solution is the antimicrobial development of the highly bioactive natural product juglone, yet few studies exist on its antibacterial mechanism against L. monocytogenes. Thus, we aimed to elucidate the antibacterial mechanism of action of juglone against L. monocytogenes by determining the resultant cell morphology, membrane permeability, membrane integrity, and proteome changes. The minimum inhibitory concentration of juglone against L. monocytogenes was 50 μg/mL, and L. monocytogenes treated with juglone had longer lag phases compared to controls. Juglone induced L. monocytogenes cell dysfunction, leakage of potassium ions, and membrane potential hyperpolarization. Confocal laser scanning microscopy and field-emission-gun scanning electron microscope assays revealed clear membrane damage due to juglone treatment. Fourier transform infrared analyses showed that L. monocytogenes responded to juglone by some conformational and compositional changes in the molecular makeup of the cell membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that juglone either destroyed proteins or inhibited proteins synthesis in L. monocytogenes. Therefore, our findings established juglone as a natural antibacterial agent with potential to control foodborne L. monocytogenes infections.

Enhanced antimicrobial, antibiofilm and ecotoxic activities of nanoencapsulated carvacrol and thymol as compared to their free counterparts

Essential oils active components emerged as captivating antimicrobials to control biofilms developed on food contact surfaces. Free and nanoencapsulated carvacrol (CAR) and thymol (THY) were assessed as antimicrobials against Salmonella Enteritidis biofilms adhered to stainless steel. The developed spherical nanocapsules using the spray-drying technique showed an average size ranged between 159.25 and 234.76 nm and zeta potential values ranged between 23.60 and 24.66 mV. The minimal inhibitory concentrations (MIC) of free THY and CAR were both 1.25 mg L−1. Nanoencapsulation reduced MIC values to 0.62 mg L−1 (THY) and 0.31 mg L−1 (CAR). Furthermore, the exposure to free and nanoencapsulated CAR and THY induced a destabilization of bacterial membranes with obvisous morphological deformations and a pronounced leakage of potassium ions and green fluorescent proteins. Eradication of S. Enteritidis biofilms developed on stainless steel was achieved following a 15 min treatment with nanoencapsulated CAR and THY at 2 MIC. Free antimicrobial exposures induced up to 4.27 log CFU cm−2 reductions. Additionally, the ecotoxicity tests against Daphnia magna crustaceans reported a non-toxicity of both free and nanoencapsulated CAR and THY after 48 h exposure. Thereby, both CAR and THY antimicrobials proved to be promising natural surface disinfectants that require further exploration and incorporation in food industries.

C. elegans monitor energy status via the AMPK pathway to trigger innate immune responses against bacterial pathogens

Pathogen recognition and the triggering of host innate immune system are critical to understanding pathogen-host interaction. Cellular surveillance systems have been identified as an important strategy for the identification of microbial infection. In the present study, using Bacillus thuringiensis-Caenorhabditis elegans as a model, we found an approach for surveillance systems to sense pathogens. We report that Bacillus thuringiensis Cry5Ba, a typical pore-forming toxin, caused mitochondrial damage and energy imbalance by triggering potassium ion leakage, instead of directly targeting mitochondria. Interestingly, we find C. elegans can monitor intracellular energy status to trigger innate immune responses via AMP-activated protein kinase (AMPK), secreting multiple effectors to defend against pathogenic attacks. Our study indicates that the imbalance of energy status is a prevalent side effect of pathogen infection. Furthermore, the AMPK-dependent surveillance system may serve as a practicable strategy for the host to recognize and defense against pathogens.

Electro-oxidation treatment of sewage water using mixed metal oxide anodes for microbial decontamination

Electrochemical technologies provide an alluring decentralized wastewater treatment system to remove microbial load from contaminated water. The mixed metal oxide (MMO) anodes used for this electrooxidation (EO) technology are stable due to the presence of titanium, ruthenium, iridium, and platinum oxides. This EO technology works by passing current in the presence of minimum electrolyte concentration, sufficient to kill the bacteria present in sewage and simulated water. This technology can be used in sewage pre-treatment plants so that the microbes present in sewage water will not come into the environment, thus, preventing the spread of an epidemic. The lab-scale electrochemical disinfection experiments were performed under batch conditions to see the inactivation efficacy of the EO system with eight different bacteria (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella enterica, Acinetobacter calcoacetius, Serratia marcescens, Listeria sp., Enterococcus faecalis). The results showed that at an optimized condition of current density (2.38 mA/cm2), NaCl concentration (1 g/300 mL), treatment time (2 min), 100% inactivation of all the eight bacteria in simulated water was achieved. Under the same optimized conditions, 100% inactivation was achieved with actual sewage water with a treatment time of 8 min. Further, potassium ion leakage and vital staining tests were performed to confirm the complete inactivation of bacteria. The electrodes used in this study were durable even after 80 cycles, as demonstrated through FE-SEM/EDS, XRD, and Raman spectra. Thus, this developed EO system with inexpensive MMO electrodes would open up new channels in treating waterborne epidemics, especially in healthcare facilities.

Inactivation of bacteria present in secondary municipal wastewater effluent using the hybrid effect of Fe–TiO2 catalyst

A low-cost waste-driven visible active iron-titanium oxide composite was fabricated for the treatment of real municipal wastewater. The structured composite was characterized using morphological and spectroscopic techniques such as FE-SEM, XRD, UV-DRS, indicating that the fabrication procedure could uniformly coat the TiO2 particles over the clay-supported composite spheres. The iron-titanium oxide composite was efficient in introducing a hybrid effect of photocatalysis and photo-Fenton that is being facilitated by the use of waste foundry sand (WFS) and waste fly ash (WFA) (both natural sources of iron) with a TiO2 layer on the outer surface. The main emphasis was given to the disinfection of bacteria along with the degradation of organic matter in terms of BOD and COD present in municipal wastewater. This hybrid technology showed 100% inactivation of bacteria with optimized parameters such as 0.9 g L−1 of H2O2 dose, 5.5 pH, and 100% surface area covered with the catalyst in 60 min of treatment time. 54% and 40% reduction in BOD and COD was also observed in 60 min. The kinetic rate constant was found to be 2 times higher in the hybrid process as compared to the individual process. The damage to the cell wall was confirmed by potassium ion leakage and FE-SEM analysis. Fe–TiO2 composites demonstrated excellent stability even after recycling 35 times, indicating the lasting durability of the composites. The findings of this study demonstrate the possibility for waste materials to be used as a viable approach for the bacterial inactivation application with the potential of the field-scale application.

Efficacy of Slightly Acidic Electrolyzed Water for Inactivation of Cronobacter sakazakii and Biofilm Cells

The disinfection efficacy and mechanism of activity of slightly acidic electrolyzed water (SAEW) were investigated against Cronobacter sakazakii. Treatment with three concentrations of SAEW decreased C. sakazakii by 23 to 55% after 2 min. Propidium iodide uptake and scanning electron micrographs indicated that SAEW treatment damaged cell integrity and changed membrane permeability resulting in leakage of nucleic acids (109.7%), intercellular proteins (692.3%), and potassium ions (53.6%). The ability to form biofilms was also reduced. SAEW treatment reduced the activity of superoxide dismutase and catalase from 100.73 and 114.18 U/mg protein to 50.03 and 50.13 U/mg protein, respectively. Expression of C. sakazakii response regulator genes (katG, rpoS, phoP, glpK, dacC, and CSK29544_RS05515) was reduced, which blocked repair of osmotic stress-induced damage and inhibited biofilm formation. These findings provide insight into the effects of SAEW on bacterial genotype and phenotype.

Oxysterols Protect Epithelial Cells Against Pore-Forming Toxins.

Many species of bacteria produce toxins such as cholesterol-dependent cytolysins that form pores in cell membranes. Membrane pores facilitate infection by releasing nutrients, delivering virulence factors, and causing lytic cell damage - cytolysis. Oxysterols are oxidized forms of cholesterol that regulate cellular cholesterol and alter immune responses to bacteria. Whether oxysterols also influence the protection of cells against pore-forming toxins is unresolved. Here we tested the hypothesis that oxysterols stimulate the intrinsic protection of epithelial cells against damage caused by cholesterol-dependent cytolysins. We treated epithelial cells with oxysterols and then challenged them with the cholesterol-dependent cytolysin, pyolysin. Treating HeLa cells with 27-hydroxycholesterol, 25-hydroxycholesterol, 7α-hydroxycholesterol, or 7β-hydroxycholesterol reduced pyolysin-induced leakage of lactate dehydrogenase and reduced pyolysin-induced cytolysis. Specifically, treatment with 10 ng/ml 27-hydroxycholesterol for 24 h reduced pyolysin-induced lactate dehydrogenase leakage by 88%, and reduced cytolysis from 74% to 1%. Treating HeLa cells with 27-hydroxycholesterol also reduced pyolysin-induced leakage of potassium ions, prevented mitogen-activated protein kinase cell stress responses, and limited alterations in the cytoskeleton. Furthermore, 27-hydroxycholesterol reduced pyolysin-induced damage in lung and liver epithelial cells, and protected against the cytolysins streptolysin O and Staphylococcus aureus α-hemolysin. Although oxysterols regulate cellular cholesterol by activating liver X receptors, cytoprotection did not depend on liver X receptors or changes in total cellular cholesterol. However, oxysterol cytoprotection was partially dependent on acyl-CoA:cholesterol acyltransferase (ACAT) reducing accessible cholesterol in cell membranes. Collectively, these findings imply that oxysterols stimulate the intrinsic protection of epithelial cells against pore-forming toxins and may help protect tissues against pathogenic bacteria.

Antibacterial mechanism of apple phloretin on physiological and morphological properties of Listeria monocytogenes

Green bactericides are excellent strategies for reducing food spoilage. Phloretin was regarded as efficient, broad-spectrum and safe preservative, which showed enormous potential in food industry. Inhibitory mechanism of phloretin on Listeria monocytogenes was systematically elucidated through integrity of cell membrane, intracellular protein content, energy level, and nucleic acid morphological character. SEM and TEM results showed minimal injury of cell membrane, which was further proved by a leakage of potassium ions, ATP and little protein and nucleic acid. There were drastic reduction of intracellular or total protein and ATP compared to cell without phloretin treatment. However, the specific activity of hexokinase and isocitrate dehydrogenase were unaffected after phloretin treatment. From these results, we concluded that phloretin’s bacteriostatic mechanism is decreasing the intracellular protein content and energy level resulting from DNA aggregation. This finding was prospective to fully illustrate the phloretin’s mechanism and develop it into a natural preservative in food industry.