Leakage Of Potassium Ions Research Articles

Synthesis and antibacterial activity studies in vitro of indirubin-3'-monoximes.

Multi-drug-resistant microbial pathogens are a serious global health problem. New compounds with antibacterial activity serve as good candidates for developing novel antibacterial drugs which is very urgent and important. In this work, based on the unique scaffold of indirubin, an active ingredient of traditional Chinese medicine formulation Danggui Luhui Wan, we synthesized 29 indirubin-3′-monoximes and preliminarily evaluated their antibacterial activities. The antibacterial activity results demonstrated that the synthesized indirubin-3′-monoximes 5a–5z and 5aa–5ad displayed good potency against S. aureus ATCC25923 (MIC = 0.4–25.6 μg mL−1). Among them, we found that the 5-F, 5-Cl and 7-CF3 substituted indirubin-3′-monoximes 5r, 5s and 5aa also showed better antibacterial efficiency for S. aureus (MICs up to 0.4 μg mL−1) than the prototype natural product indirubin (MIC = 32 μg mL−1). More importantly, indirubin-3′-monoxime 5aa has certain synergistic effect with levofloxacin against clinic multidrug-resistant S. aureus (fractional inhibitory concentration index: 0.375). In addition, relevant experiments including electron microscopy observations, PI staining and the leakage of extracellular potassium ions and nucleic acid (260 nm) have been performed after treating S. aureus with indirubin-3′-monoxime 5aa, and the results revealed that indirubin-3′-monoximes could increase the cell membrane permeability of S. aureus. Although indirubin-3′-monoxime 5aa showed some cytotoxicity toward SH-SY5Y cells relative to compounds 5r and 5s, the skin irritation test of male mice after shaving showed that compound 5aa at a concentration of 12.8 μg mL−1 had no toxicity to mouse skin, and it could be used as a leading compound for skin antibacterial drugs.

Imaging of Potassium and Calcium Distribution in Plant Tissues and Cells to Monitor Stress Response and Programmed Cell Death.

In plants, the response to stress, such as salinity, pathogen attack, drought, high concentration of metals, hyperthermia, and hypothermia, is usually accompanied by potassium ion (K+) leakage from the cytosol to the cell wall, mediated by plasma membrane cation conductivity. Stress-induced electrolyte leakage co-occurs with accumulation of reactive oxygen species (ROS) and calcium ions (Ca2+) and often results in programmed cell death (PCD). The development of X-ray and mass spectrometry (MS) based imaging techniques has enabled insight into the spatial tissue and cell-specific redistribution of major and trace elements during the stress response. In this chapter a workflow for sample preparation, imaging, and image analysis by X-ray and MS based techniques is presented.

Carvacrol and Thymol Combat Desiccation Resistance Mechanisms in Salmonella enterica Serovar Tennessee.

Some Salmonella enterica serovars are frequently associated with disease outbreaks in low-moisture foods (LMF) due to their ability to adapt efficiently to desiccation stress. These serovars are often persistent during food processing. Disruption of these resistance responses was accomplished previously using the membrane-active lipopeptide, paenibacterin. This study was initiated to determine how desiccation resistance mechanisms are overcome when Salmonella Tennessee, a known resistant serovar, is treated with the membrane-active food additives carvacrol and thymol. Knowing that the minimum inhibitory concentrations (MICs) of carvacrol and thymol against Salmonella Tennessee are 200 and 100 µg/mL, the concentrations tested were 100–400 and 50–200 µg/mL, respectively. Results show that desiccation-adapted Salmonella Tennessee, prepared by air drying at 40% relative humidity and 22–25 °C for 24 h, was not inactivated when exposed for 4.0 h to less than 2xMIC of the two additives. Additionally, treatment of desiccation-adapted Salmonella Tennessee for 120 min with carvacrol and thymol at the MIC-level sensitized the cells (1.4–1.5 log CFU/mL reduction) to further desiccation stress. Treating desiccation-adapted Salmonella Tennessee with carvacrol and thymol induced leakage of intracellular potassium ions, reduced the biosynthesis of the osmoprotectant trehalose, reduced respiratory activity, decreased ATP production, and caused leakage of intracellular proteins and nucleic acids. Carvacrol, at 200–400 µg/mL, significantly downregulated the transcription of desiccation-related genes (proV, STM1494, and kdpA) as determined by the reverse-transcription quantitative PCR. The current study revealed some of the mechanisms by which carvacrol and thymol combat desiccation-resistant Salmonella Tennessee, raising the feasibility of using these additives to control desiccation-adapted S. enterica in LMF.

Purification and Characterization of Plantaricin YKX and Assessment of Its Inhibitory Activity Against Alicyclobacillus spp.

Consumers prefer natural over synthetic chemical preservatives on a food label. Therefore, it is crucial to ensure the safety and efficacy of such natural preservatives. The emergence of heat-resistant spore-forming Alicyclobacillus spp. has been associated with spoilage problems in the fruit juice industry. Herein, a bacteriocin-producing stain YKX was isolated from the traditional pickles in Hanzhong City, China, and it was identified as Lactobacillus plantarum by morphological, biochemical, physiological, and genotypic features. A stable bacteriocin, plantaricin YKX, was isolated, purified, and tested for its efficacy against Alicyclobacillus acidoterrestris. Plantaricin YKX is a 14-amino acid peptide (Lys-Tyr-Gly-Asn-Gly-Leu-Ser-Arg-Ile-Phe-Ser-Ala-Leu-Lys). Its minimal inhibitory concentrations (MICs) against the tested bacterial and fungal strains were ranged from 16 to 64 μg/mL. It is thermostable and active at pH 3–8. The flow cytometry data and microscopic observations suggested that plantaricin YKX can augment cell membrane permeability, induce potassium ion leakage and pore formation, and disrupt cell membranes. It also affects spore germination and guaiacol production of A. acidoterrestris, probably due to upregulation of the luxS gene linked to quorum sensing.

Structural elucidation, mechanism of action, antibacterial proficiency and synergistic forte of purified Euphorbia hirta whole plant extract against multi-drug resistant otitis media infection

This study investigated the antibacterial potential of Euphorbia hirta whole plant extracts, honey and conventional antibiotics and their synergistic effects against selected multidrug resistant and typed bacterial strains associated with otitis media. E. hirta whole plant extract was purified using column chromatography technique. The antibacterial assays of extracts were done using standard microbiological procedures. Protein, sodium and potassium ion leakage of the synergistic mixtures was determined using flame-photometry. At 100 mg/ml, acetone extracts presented highest inhibition against S. aureus (NCTC 6571) with 32 ± 0.83 mm zone of inhibition. The fractional inhibitory concentration indices displayed higher synergism in combination of plant extract, honey and ciprofloxacin against P. mirabilis at 0.02 compared to drug combination synergy standard (≤ 0.5). This work revealed augmentation of ciprofloxacin potency when combined with purified E. hirta acetone extract and honey and implies their high potential in the treatment of multidrug resistant infection of otitis media.

Potassium ion leakage impairs thermotolerance in Corynebacterium glutamicum

Corynebacterium glutamicum, a gram-positive bacterium, can produce amino acids such as glutamic acid and lysine. The heat generated during cell growth and/or glutamate fermentation disturbs both the cell growth and fermentation. To overcome such a negative effect of the fermentation heat, we have tried to establish a high temperature fermentation. One of the approach is to create a thermotolerant strains, while the other is to create an optimum culture conditions able for the strain to grow at higher temperatures. In this study, we focused on the latter approach, where we examined the effect of potassium ion on cell growth at high growth temperatures of C.glutamicum. The supplementation of high concentrations of potassium chloride (300mM) (or sorbitol, an osmolyte) mitigated the repressed cell growth induced by high temperature at 39°C or 40°C. The intracellular potassium concentration declines from 300mM to ∼150mM by increasing the growth temperature but not by supplementing potassium chloride or sorbitol. Furthermore, invitro experiments revealed that the potassium ion leakage occurs at high temperatures, which was mitigated in the presence of high concentrations of extracellular potassium chloride. This suggested that the presence of high osmolyte in the culture medium could inhibit the potassium ion leakage induced by high temperature and subsequently support cell growth at high temperatures.

Effects of Antibacterial Peptide F1 on Bacterial Liposome Membrane Integrity.

Previous studies from our lab have shown that the antimicrobial peptide F1 obtained from the milk fermentation by Lactobacillus paracasei FX-6 derived from Tibetan kefir was different from common antimicrobial peptides; specifically, F1 simultaneously inhibited the growth of Gram-negative and Gram-positive bacteria. Here, we present follow-on work demonstrating that after the antimicrobial peptide F1 acts on either Escherichia coli ATCC 25922 (E. coli) or Staphylococcus aureus ATCC 63589 (S. aureus), their respective bacterial membranes were severely deformed. This deformation allowed leakage of potassium and magnesium ions from the bacterial membrane. The interaction between the antimicrobial peptide F1 and the bacterial membrane was further explored by artificially simulating the bacterial phospholipid membranes and then extracting them. The study results indicated that after the antimicrobial peptide F1 interacted with the bacterial membranes caused significant calcein leakage that had been simulated by different liposomes. Furthermore, transmission electron microscopy observations revealed that the phospholipid membrane structure was destroyed and the liposomes presented aggregation and precipitation. Quartz Crystal Microbalance with Dissipation (QCM-D) results showed that the antimicrobial peptide F1 significantly reduced the quality of liposome membrane and increased their viscoelasticity. Based on the study's findings, the phospholipid membrane particle size was significantly increased, indicating that the antimicrobial peptide F1 had a direct effect on the phospholipid membrane. Conclusively, the antimicrobial peptide F1 destroyed the membrane structure of both Gram-negative and Gram-positive bacteria by destroying the shared components of their respective phospholipid membranes which resulted in leakage of cell contents and subsequently cell death.

Green synthesis of walnut shell hydrochar, its antimicrobial activity and mechanism on some pathogens as a natural sanitizer

Hydrochar of waste walnut shells (WSH) was synthesized in the eco-friendly subcritical water medium (SWM) and its potential to fight against Klebsiella pneumoniae (K. pneumoniae), Staphylococcus aureus (S. aureus), Candida albicans (C. albicans) and Candida parapsilosis (C. parapsilosis) was investigated. Minimum Inhibitory Concentration (MIC) values of the WSH were 3.01 g/mL, 2.06 g/mL, 1.95 g/mL, and 3.12 g/mL for K. pneumoniae, S. aureus, C. albicans and C. parapsilosis, respectively. Survival of the pathogens was investigated by 3 min surface disinfection test exposure to WSH. While the highest inhibition was seen for C. parapsilosis (96.67%) on paper surface with 0.3 g/mL of bovine serum albumin (BSA), the lowest inhibition was determined for C. albicans (6.44%) on the plastic glass surface with 3 g/mL of BSA. An increase in protein, DNA, and potassium ion (K+) leakage was observed after microorganisms were incubated with WSH. This study provided an experimental basis for the practical application of WSH as a natural sanitizer agent.

Antibacterial characteristics and mechanisms of action of Aronia melanocarpa anthocyanins against Escherichia coli

This study aimed to evaluate the antibacterial activity and mechanism of action of Aronia melanocarpa anthocyanins (AMAs) against Escherichia coli. AMAs displayed strong antibacterial activity against Escherichia coli. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AMAs against E. coli was 0.625 mg/mL and 1.25 mg/mL, respectively. Analysis of alkaline phosphatase activity, potassium ion leakage, absorption material release at 260 nm, and change in soluble protein content revealed that AMAs could destroy the cell wall and membrane structure of E. coli. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) further confirmed the cell wall and cell membrane damage induced by AMAs. In addition, the interaction between AMAs and DNA was elucidated through agarose gel retardation and fluorescence spectroscopy. In summary, the present results indicate that AMAs kill E. coli by attacking multiple targets in the bacterial cell, which could favour the application of AMAs as natural food preservatives.

Effect of naturally isolated hydroquinone in disturbing the cell membrane integrity of Pseudomonas aeruginosa MTCC 741 and Staphylococcus aureus MTCC 740

Nosocomial pathogens cause various health problems in human and many novel drugs are under investigation to combat the pathogens. The present study explains the naturally derived hydroquinone possible mode of action against Pseudomonas aeruginosa MTCC 741 and Staphylococcus aureus MTCC 740. Time kill studies, cell viability assays, membrane potential assays, and potassium release assays were carried out to study the mode of action. Time kill studies revealed the rapid death of bacterial pathogens exposed to 4X MIC (Minimum inhibitory concentration) of the hydroquinone. Cell viability assay results showed that nearly half of the cell destruction of test pathogens occurred within one hour. Transmission electron microscopic (TEM) observations revealed the disruption of the cell membrane, which caused severe ultrastructural changes in both test pathogens. Hydroquinone dissipated the membrane potential of test pathogens, as confirmed by the depolarization of membrane potential, increases in permeability and leakage of intracellular potassium ions. At the concentration of 2X MIC hydroquinone in 5 min, about 91.41% and 84.85% potassium ions were released from P. aeruginosa MTCC 741 and S. aureus MTCC 740, respectively. This is the first report on the mode of action of naturally derived hydroquinone against clinical pathogens.

Biofilm eradication and antifungal mechanism of action against Candida albicans of cationic dicephalic surfactants with a labile linker

Our research aims to expand the knowledge on relationships between the structure of cationic dicephalic surfactants—N,N-bis[3,3_-(dimethylamine)propyl]alkylamide dihydrochlorides and N,N-bis[3,3_-(trimethylammonio)propyl]alkylamide dibromides (alkyl: n-C9H19, n-C11H23, n-C13H27, n-C15H31)—and their antifungal mechanism of action on Candida albicans. The mentioned groups of amphiphilic substances are characterized by the presence of a weak, hydrochloride cationic center readily undergoing deprotonation, as well as a stable, strong quaternary ammonium group and alkyl chains capable of strong interactions with fungal cells. Strong fungicidal properties and the role in creation and eradication of biofilm of those compounds were discussed in our earlier works, yet their mechanism of action remained unclear. It was shown that investigated surfactants induce strong oxidative stress and cause increase in cell membrane permeability without compromising its continuity, as indicated by increased potassium ion (K+) leakage. Thus experiments carried out on the investigated opportunistic pathogen indicate that the mechanism of action of the researched surfactants is different than in the case of the majority of known surfactants. Results presented in this paper significantly broaden the understanding on multifunctional cationic surfactants and their mechanism of action, as well as suggest their possible future applications as surface coating antiadhesives, fungicides and antibiofilm agents in medicine or industry.

Antifungal activity and mechanism of action of dichloromethane extract fraction A from Streptomyces libani against Aspergillus fumigatus.

This study aimed to investigate the mechanism of antifungal action of Streptomyces libani dichloromethane extract fraction A (DCEFA) against Aspergillus fumigatus and the host cytotoxicity. DCEFA was purified from S. libani by autobiography and showed strong antifungal activity against A. fumigatus. A combination of electron microscopy, cell permeability assays, total oxidant status (TOS) assay, cell cytotoxicity assay and haemolysis activity was carried out to determine the target site of DCEFA. Exposure of A. fumigatus to DCEFA caused the damage to membranous cellular structures and increased release of cellular materials, potassium ions and TOS production. DCEFA was bound to ergosterol but did not affect fungal cell wall and ergosterol content. DCEFA did not show any obvious haemolytic activity for RBCs and toxicity against HEK-293 cell line. DCEFA may inhibit A. fumigatus growth by targeting fungal cell membrane which results in the leakage of potassium ions and other cellular components, TOS production and final cell death. DCEFA of S. libani could be considered as a potential source of novel antifungals which may be useful for drug development against A. fumigatus as a life-threatening human pathogen.

Efficacy of short-synthetic antifungal peptides on pathogenic Aspergillus flavus

The efficacies of three short synthetic antifungal peptides were tested for their inhibitory action on pathogenic fungi, Aspergillus flavus. The sequences of the short synthetic peptides are PPD1- FRLHF, 66-10-FRLKFH, 77–3- FRLKFHF, respectively. These test peptides inhibited fungal growth and showed a membranolytic activity. The fungal biomass and ergosterol levels were significantly low in peptides treated samples. Further, the fungal cell wall component chitin was also found to be lower in peptides treated samples. Scanning electron microscopic images also showed highly wrinkled fungal mycelia. Significant membrane permeabilisation as well as potassium ion leakage was also observed in fungal samples treated with peptides. To assess the membrane damage, the uptake of Sytox green dye was employed. At tested concentration, peptides induced fungal membrane damage as evidenced by the green fluorescence. Further, at tested concentration, these peptides induced an oxidative stress in A.flavus as evidenced by an increase in the ROS production, malondialdehyde levels, increase in the antioxidant enzymes - superoxide dismutase, catalase with concomitant decrease in the reduced glutathione content. Additionally, a growth dependent reduction in aflatoxin levels were also observed in peptides treated samples. Docking studies on the interaction of the peptides with a trans-membrane protein calcium ATPase of A. flavus showed that all the peptides were able to bind to the protein with high z rank score. The activity of the calcium ATPase was significantly decreased in peptides treated fungal samples, thereby validating the docking results. Among all the tested peptides, 77–3 peptide exhibited the maximal membrane damage property.

Natural Antimicrobials Suitable for Combating Desiccation-Resistant Salmonella enterica in Milk Powder.

Some Salmonella enterica strains survive well in low-water activity (low-aw) foods and cause frequent salmonellosis outbreaks in these products. Methods are needed to overcome such desiccation-resistant Salmonella and to improve the safety of low-aw foods. Building on a recent finding, we hypothesized that natural antimicrobial food additives, which are active against cytoplasmic membrane, could overcome this desiccation resistance phenomenon, and thus, sensitize the pathogen to drying and mild processing. Food additives were screened for the ability to cause leakage of intracellular potassium ions; retention of these ions is vital for protecting Salmonella against desiccation. Two antimicrobial food additives, carvacrol and thymol, caused considerable potassium leakage from the desiccation-resistant S. enterica serovars, Tennessee and Livingstone. Thus, carvacrol and thymol were investigated for their ability to sensitize the desiccation-adapted S. enterica to heat treatment. The combined use of food additives, at their minimum inhibitory concentrations, with heat treatment at 55 °C for 15 min caused 3.1 ± 0.21 to more than 5.5 log colony forming unit (CFU)/mL reduction in desiccation-adapted S. enterica, compared to 2.4 ± 0.53–3.2 ± 0.11 log CFU/mL reduction by sole heat treatment. Carvacrol was the additive that caused the greatest potassium leakage and sensitization of Salmonella to heat; hence, the application of this compound was investigated in a food model against Salmonella Typhimurium ASD200. Addition of carvacrol at 200 or 500 ppm into liquid milk followed by spray-drying reduced the strain’s population by 0.9 ± 0.02 and 1.3 ± 0.1 log CFU/g, respectively, compared to 0.6 ± 0.02 log CFU/g reduction for non-treated spray-dried milk. Additionally, freeze-drying of milk treated with high levels of carvacrol (5000 ppm) reduced the population of Salmonella Typhimurium ASD200 by more than 4.5 log CFU/g, compared to 1.1 ± 0.4 log CFU/g reduction for the freeze-dried untreated milk. These findings suggest that carvacrol can combat desiccation-resistant S. enterica, and thus, potentially improve the safety of low-aw foods.

Antibacterial and Antidiarrheal Activities of Houttuynia Cordata Thunb. And Portulaca Oleracea L. Extracts Against Enterotoxigenic Escherichia Coli

Houttuynia cordata Thunb. and Portulaca oleracea L. have been used as medicinal and food homologous plants. This research focused on evaluating the possibilities of the combination of H. cordata and P. oleracea extracts as alternatives to antibiotics. The antimicrobial and antidiarrheal activities of the combination of ethanol extract and essential oil from H. cordata (HEE and HEO) and ethanol extract from P. oleracea (PEE) against enterotoxigenic Escherichia coli were studied. Results showed the combination (CB-HHP) of HEE, HEO, and PEE indicated synergistic effect on E. coli , and the concentration consist when HEE, HEO, and PEE were at a concentration of 1, 0.56, and 0.5 mg/mL, respectively, was regarded as minimal inhibitory concentration (MIC) of CB-HHP. CB-HHP increased the permeability of cell wall and cell membrane and affected the integrity of cell membrane, as evidenced by the leakage of alkaline phosphatase (AKP), potassium ion (K+), and protein, and changes in cell morphology further proved the damage to the cell wall and membrane of E. coli by CB-HHP. In addition, CB-HHP treatment significantly inhibited formation of E. coli bacterial biofilm. In vivo, gavaging with CB-HHP (HEE:HEO:PEE = 1:0.56:0.5, 800 mg/kg body weight) could significantly relieve the weight loss and diarrhea rate and protect the duodenal villi of mice infected by E. coli , as well as increasing the concentration of immunoglobulin (Ig) G, IgM, and IgA in serum. Therefore, CB-HHP was demonstrated remarkable antibacterial activity, antidiarrheal activity, and intestinal protective function, with potential to be used as food-derived additives.

Study on antimicrobial and antioxidant potentials of Alchornea cordifolia (Linn.): an in vitro assessment

This study investigated antimicrobial properties of Alchornea cordifolia leaf extract against a panel of selected bacterial strains. It also assessed antioxidant and phytochemical properties of the plant constituents responsible for the bioactive actions recorded. The antimicrobial potentials of the crude extract along with the fractions obtained were determined using the agar well-diffusion method. The most active fraction was partially purified by a combination of thin-layer and column chromatography; and the antimicrobial activity of the resulting samples was tested against the bacterial strains. The results showed that the leaf extract of A. cordifolia and the obtained fractions exhibited varying degrees of antibacterial activities. Phytochemical tests revealed the presence of alkaloids, flavonoids, tannins, saponins, phenols, resins, sterols, carbohydrates and terpenoids. The minimum inhibitory concentrations of the crude extracts were 0.55–4.38 mg/mL while those of the chloroform and ethyl acetate fractions were 0.08–2.50 and 0.08–0.63 mg/mL, respectively. The rate of kill assay showed that the percentage of cells killed was related to concentrations and contact time. The nucleotide and potassium ion leakage from test cells also increased with an increase in the fraction concentrations as well as the contact time intervals. Alchornea cordifolia extract exhibited 50% 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities at 2.1 µg/mL, whereas the ascorbic acid used as standard had a half maximal inhibitory concentration (IC50) of 12.3 µg/mL. The major active constituent of the purified sample was 6-octadecanoic acid. This study showed that A. cordifolia leaf extract, which possesses antioxidant properties, exhibited significant antimicrobial activities against the test pathogens.

An Enhanced Variant Designed From DLP4 Cationic Peptide Against Staphylococcus aureus CVCC 546.

Insect defensins are promising candidates for the development of potent antimicrobials against antibiotic-resistant Staphylococcus aureus (S. aureus). An insect defensin, DLP4, isolated from the hemolymph of Hermetia illucens larvae, showed low antimicrobial activity against Gram-positive (G+) pathogens and high cytotoxicity, which limited its effective therapeutic application. To obtain more potent and low cytotoxicity molecules, a series of peptides was designed based on the DLP4 template by changing the conservative site, secondary structure, charge, or hydrophobicity. Among them, a variant designated as ID13 exhibited strong antibacterial activity at low MIC values of 4–8 μg/mL to G+ pathogens (S. aureus: 4 μg/mL; Staphylococcus epidermidis: 8 μg/mL; Streptococcus pneumoniae: 4 μg/mL; Streptococcus suis: 4 μg/mL), which were lower than those of DLP4 (S. aureus: 16 μg/mL; S. epidermidis: 64 μg/mL; S. pneumoniae: 32 μg/mL; S. suis: 16 μg/mL), and cytotoxicity of ID13 (71.4% viability) was less than that of DLP4 (63.8% viability). ID13 could penetrate and destroy the cell membrane of S. aureus CVCC 546, resulting in an increase in potassium ion leakage; it bound to genomic DNA (gDNA) and led to the change of gDNA conformation. After treatment with ID13, perforated, wrinkled, and collapsed S. aureus CVCC 546 cells were observed in electron microscopy. Additionally, ID13 killed over 99.99% of S. aureus within 1 h, 2 × MIC of ID13 induced a post-antibiotic effect (PAE) of 12.78 ± 0.28 h, and 10 mg/kg ID13 caused a 1.8 log10 (CFU/g) (CFU: colony-forming units) reduction of S. aureus in infected mouse thigh muscles and a downregulation of TNF-α, IL-6, and IL-10 levels, which were superior to those of DLP4 or vancomycin. These findings indicate that ID13 may be a promising peptide antimicrobial agent for therapeutic application.

Effect of Achillea santolina essential oil on bacterial biofilm and its mode of action

Abstract Increased multidrug resistance prompted researchers to search for a new drug that has the ability to overcome antibiotic resistant pathogens. Essential oils have been used in folk medicine for centuries, therefore, they could be employed as an effective alternative to antibiotics without having secondary side effects. The aim of the present study was to test the antibacterial and antibiofilm activity of the essential oil of Achillea santolina and to ascertain its mode of action. Minimum Biofilm Inhibitory Concentration (MBIC) susceptibility assays were performed using a biofilm inoculator with a 96-well plate with peg led. Minimum Inhibitory Concentration (MIC) was performed in normal microtitre plates using a twofold dilution series. Achillea santolina essential oil (ASEO) was able to overcome the resistance of all tested bacteria. The MIC values were in the range of 250-1000 µg/ml, while the MBC values were in the range of 500-2000 µg/ml. ASEO increased leakage of potassium ions from the cell membrane and increased release of cellular materials – suggesting that the cell membrane is the target and site of action of ASEO. Moreover, ASEO was able to inhibit initial adherence of methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300) at sub-inhibitory concentrations through alterations to cell membrane.

Effects of CGA-N12 on the membrane structure of Candida tropicalis cells.

The antimicrobial peptide CGA-N12 (NH2-ALQGAKERAHQQ-COOH) is an active peptide derived from chromogranin A (CGA) and consists of the 65th to 76th amino acids of the N-terminus. The results of our previous studies showed that CGA-N12 exerts anti-Candida activity by inducing apoptosis without destroying the integrity of cell membranes. In this study, the effect of CGA-N12 on the cell membrane structure of Candida tropicalis was investigated. CGA-N12 resulted in the dissipation of the membrane potential, the increase in membrane fluidity, and the outflow of potassium ions in C. tropicalis without significantly changing the ergosterol level. Fluorescence quenching was applied to evaluate the membrane channel characteristics induced by CGA-N12 through detection of the following: membrane permeability of hydrated Cl- (ϕ ≈ 0.66 nm) using the membrane-impermeable halogen anion-selective fluorescent dye lucigenin, passage of the membrane-impermeable dye carboxyfluorescein (CF) (ϕ ≈ 1 nm) through the membrane, and membrane permeation of H3O+ based on the membrane non-permeable pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS). In conclusion, CGA-N12 can induce the formation of non-selective ion channels <1 nm in diameter in the membranes of C. tropicalis, resulting in the leakage of potassium ions, chloride ions, and protons, among others, leading to dissipation of the membrane potential. As a result, the fluidity of membranes is increased without destroying the synthesis of ergosterol is not affected.

Antibiotic activities of the natural antimicrobial substance produced by Lactobacillus paracasei FX-6 against Pseudomonas putida

In our previous study, Lactobacillus paracasei FX-6 was isolated from kefir, and the antimicrobial substance produced by FX-6 had broad spectrum antimicrobial activity, which had a good effect of preservation on chicken breast and could significantly inhibit the dominant spoiled bacteria Pseudomonas. In this study, we isolated and identified the dominant bacteria Pseudomonas, and we also researched the antibacterial effect and mechanism of antibacterial substance against Pseudomonas. The dominant bacteria Pseudomonas was isolated from spoiled chicken breast meat and identified as Pseudomonas putida. The result showed that the minimum inhibitory concentration (MIC) of antibacterial substance against Pseudomonas putida was 12.5 mg/mL. The Propidium iodide (PI) infiltration experiments and leakage of potassium ions indicated the antimicrobial substance could increase the permeability of the bacterial cell membrane. Scanning electron microscope (SEM) revealed that the antimicrobial substance damaged the morphology of Pseudomonas putida cell. Result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that the synthesis of protein of Pseudomonas putida could be affected by the antibacterial substance. The result of agarose gel retardation indicated that the genomic DNA could be combined with the antimicrobial substance.