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Bacterial inactivation by 405 nm light is accredited to the photoexcitation of intracellular porphyrin molecules resulting in energy transfer and the generation of reactive oxygen species that impart cellular oxidative damage. The specific mechanism of cellular damage, however, is not fully understood. Previous work has suggested that destruction of nucleic acids may be responsible for inactivation; however, microscopic imaging has suggested membrane damage as a major constituent of cellular inactivation. This study investigates the membrane integrity of Escherichia coli and Staphylococcus aureus exposed to 405 nm light. Results indicated membrane damage to both species, with loss of salt and bile tolerance by S. aureus and E. coli, respectively, consistent with reduced membrane integrity. Increased nucleic acid release was also demonstrated in 405 nm light-exposed cells, with up to 50 % increase in DNA concentration into the extracellular media in the case of both organisms. SYTOX green fluorometric analysis, however, demonstrated contradictory results between the two test species. With E. coli, increasing permeation of SYTOX green was observed following increased exposure, with >500 % increase in fluorescence, whereas no increase was observed with S. aureus. Overall, this study has provided good evidence that 405 nm light exposure causes loss of bacterial membrane integrity in E. coli, but the results with S. aureus are more difficult to explain. Further work is required to gain greater understanding of the inactivation mechanism in different bacterial species, as there are likely to be other targets within the cell that are also impaired by the oxidative damage from photo-generated reactive oxygen species.

Atmospheric cold plasma (ACP) is a promising nonthermal technology effective against a wide range of pathogenic microorganisms. Reactive oxygen species (ROS) play a crucial inactivation role when air or other oxygen-containing gases are used. With strong oxidative stress, cells can be damaged by lipid peroxidation, enzyme inactivation, and DNA cleavage. Identification of ROS and an understanding of their role are important for advancing ACP applications for a range of complex microbiological issues. In this study, the inactivation efficacy of in-package high-voltage (80 kV [root mean square]) ACP (HVACP) and the role of intracellular ROS were investigated. Two mechanisms of inactivation were observed in which reactive species were found to either react primarily with the cell envelope or damage intracellular components. Escherichia coli was inactivated mainly by cell leakage and low-level DNA damage. Conversely, Staphylococcus aureus was mainly inactivated by intracellular damage, with significantly higher levels of intracellular ROS observed and little envelope damage. However, for both bacteria studied, increasing treatment time had a positive effect on the intracellular ROS levels generated.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major problem in clinical settings, and because it is resistant to most antimicrobial agents, MRSA infections are difficult to treat. We previously reported that synthetic macrocyclic bis(bibenzyl) derivatives, which were originally discovered in liverworts, had anti-MRSA activity. However, the action mechanism responsible was unclear. In the present study, we elucidated the action mechanism of macrocyclic bis(bibenzyl) RC-112 and its partial structure, IDPO-9 (2-phenoxyphenol). Survival experiments demonstrated that RC-112 had a bactericidal effect on MRSA, whereas IDPO-9 had bacteriostatic effects. IDPO-9-resistant mutants exhibited cross-resistance to triclosan, but not to RC-112. The mutation was identified in the fabI, enoyl-acyl carrier protein reductase gene, a target of triclosan. We have not yet isolated the RC-112-resistant mutant. On the other hand, the addition of RC-112, unlike IDPO-9, caused the inflow of ethidium and propidium into S. aureus cells. RC-112-dependent ethidium outflow was observed in ethidium-loaded S. aureus cells. Transmission electron microscopy also revealed that S. aureus cells treated with RC-112 had intracellular lamellar mesosomal-like structures. Intracellular Na+ and K+ concentrations were significantly changed by the RC-112 treatment. These results indicated that RC-112 increased membrane permeability to ethidium, propidium, Na+, and K+, and also that the action mechanism of IDPO-9 was different from those of the other compounds.

UNLABELLED This study evaluated the effect of soy lecithin on the bile resistance of Lactobacillus plantarum. Six strains were cultured in MRS broth supplemented with soy lecithin at different concentrations. The strains incubated in MRS broth with 1·0% soy lecithin showed no inhibitory effect on cell growth. After culturing in MRS broth with 0·2-1·0% soy lecithin, the survival rate of harvested cells increased significantly (P < 0·05) in the 0·3% bile challenge compared with the no added soy lecithin group. The cells incubated with 0·6% soy lecithin were able to grow in an MRS broth with a higher bile salt content. The surface hydrophobicity and cell leakage in the bile challenge were assessed to reveal the physical changes caused by the addition of soy lecithin. The cell surface hydrophobicity was enhanced and the membrane integrity in the bile challenge increased after culturing with soy lecithin. A shift in the fatty acid composition was also observed, illustrating the cell membrane change in the soy lecithin culture. SIGNIFICANCE AND IMPACT OF THE STUDY In this study, we report for the first time the beneficial effect of adding soy lecithin to an MRS broth on subsequent bile tolerance of Lactobacillus plantarum. Soy lecithin had no inhibitory effect on strain viability but significantly enhanced bile resistance. Surface hydrophobicity and cell integrity increased in strains cultured with soy lecithin. The observed shift in the cell fatty acid composition indicated changes to the cell membrane. As soy lecithin is safe for use in the food industry, its protective effects can be harnessed for the development of bile-sensitive strains with health-benefit functions for use in probiotic products.

Chemical encapsulation of microbes in threedimensional polymeric microcapsules promises various applications, such as cell therapy and biosensors, and provides a basic platform for studying microbial communications. However, the cytoprotection of microbes in the microcapsules against external aggressors has been a major challenge in the field of microbial microencapsulation, because ionotropic hydrogels widely used for microencapsulation swell uncontrollably, and are physicochemically labile. Herein, we developed a simple polydopamine coating for obtaining cytoprotective capability of the alginate capsule that encapsulated Saccharomyces cerevisiae. The resulting alginate/ polydopamine core/shell capsule was mechanically tough, prevented gel swelling and cell leakage, and increased resistance against enzymatic attack and UV-C irradiation. We believe that this multifunctional core/shell structure will provide a practical tool for manipulating microorganisms inside the microcapsules.

Dialkyl-alginate biosurfactant is an amphifilik rhamnolipid biosurfactant that has the potential to be developed into an antibacterial agent. The purpose of this study was to prove that the biosurfactant of dialkyl alginate both before and after creams has antibacterial activity especially against Staphylococcus aureus and Pseudomonas aeruginosa . Test of antibacterial activity of biosurfactant dialkyl alginate at concentrations of 5%, 10% and 20% to Staphylococcus aureus and Pseudomonas aeruginosa using Hammond, et al (2011) modified quantitative method. Enbatic® 1% is used as a positive control and sterile aquadest as a negative control. Determination of antibacterial activity of dialkyl alginate biosurfactant followed by analysis of leakage of protein and nucleic acids using UV-vis Spectrophotometry and leakage of Ca 2 + and K + metal ions using Atomic Absorption Spetrophotometry (AAS). The most active concentration was formulated into cream and then performed physical evaluation (organoleptic, pH, adhesion, spreading and viscosity) and tested antibacterial activity using the well method. The test results showed that the biosurfactants of dialkyl-alginate before and after the cream was treated as antibacterial activity. The concentration of 10% was the most active concentration having activity which did not differ significantly to positive control with p value of 0,05

With CO2 and N2 as the pressure media, the effects of the moderate pressure (0.1–1.0MPa) and the holding time on the conductivities of the cell suspension of Saccharomyces cerevisiae CICC1447 and Saccharomyces cerevisiae CICC1339, as well as the absorbances of the supernatant (after centrifuged) at 280 nm (A280) and 260 nm (A260) were determined. The membrane permeability of Saccharomyces cerevisiae CICC1447 increased significantly and the cell leakage was aggravated with the pressure increase. For Saccharomyces cerevisiae CICC1339, the conductivity of the cell suspension, A280 and A260 of the supernatant fluctuated with the pressure increase; as a whole, they increased with pressure. Different from high pressure, a moderate pressure not only remarkably improved the permeability of the yeast cell membrane, but also kept yeast cell viability; moreover, the integrity of the yeast cell membrane could be maintained.

A cell immobilization technique to prevent cell leakage from the matrix in a very common system, alginate-Saccharomyces cerevisiae, was investigated. A double coating of immobilized cell beads prev...

Cells of Serratia marcescens , immobilized by entrapment in calcium alginate beads, were cultivated in two different media: a dairy waste (whey) and a complex medium (TYE). A study of the stabilization of the support employed was carried out after establishing the relationship existing between the degree of leakage from the support and the internal biomass, as well as the possible interactions between the substrate and the gel. In order to ascertain these facts, visual methods had to be developed to evaluate the number of breakages and relate these with the culture characteristics. Finally, repeated cycle cultures were carried out, establishing the evolution of product yields and the number of breakages on the support surface, showing the behaviour on whey in relation to the complex medium.

Introduction. Previously we proposed a cellular imaging technique to determine the surviving fraction of transplanted cells in vivo. Epicardial kinetics using Indium-111 determined the Debris Impulse Response Function (DIRF) and leakage coefficient parameters. Convolution-based modeling which corrected for these signal contributions indicated that 111In activity was quantitative of cell viability with half-lives within 20 hrs to 37 days. We determine if the 37-day upper limit remains valid for endocardial injections by comparing previous epicardial cell leakage parameter estimates to those for endocardial cells. Methods. Normal canine myocardium was injected (111In-tropolone) epicardially (9 injections) or endocardially (10 injections). Continuous whole body and SPECT scans for 5 hours were acquired with three weekly follow-up imaging sessions up to 20–26 days. Time-activity curves evaluated each injection type. Results. The epicardial and endocardial kinetics were not significantly different (Epi: 1286±253; Endo: 1567±470 hours P=.62). Conclusion. The original epicardial estimate of leakage kinetics has been validated for use in endocardial injections.

The efficacy of agarose in preventing VX2 carcinoma cell leakage was evaluoted and the results were compared with two traditional methods. Forty-five rabbits were divided into 3 groups: Group 1, VX2 tumor cells were injected directly into the liver and no special procedure after removal of the needle; Group 2, the puncture site was gently compressed, using an alcoholic cotton gauze, for three minutes; Group 3, 0.2 ml of heated liquid agarose was injected to seal the aperture after injection of VX2 cells. The leakage rates were 80%, 53.3% and 6.6% for group 1, group 2 and group 3, respectively. We consider agarose is a useful material in preventing the leakage in the establishment of VX2 liver tumor models.

Introduction Stem cells possess great potential in treating disk degeneration. MSC is the most clinically relevant cell source but its functional outcomes are still far from satisfaction. One of the primary reasons for the suboptimal performance of MSCs is that the majority of injected cells leaked out from the injection path. Existing attempts addressing this issue focus on further increasing the viscosity of carrier and use of BioGlue. Nevertheless, from others and our own experiences, the high intradiscal pressure in the IVD would extrude the injected cells out immediately even with solid carriers while the blocking effect of glues is only transient as large osteophyte structure was still seen in 6 months time. As a result, we hypothesize that effectively blocking the injection portal with an injectable biomaterial-based annulus plug will solve the cell leakage problem. Our group has previously developed a photochemical cross-linking technology to improve the physicochemical properties including mechanical strength, chemical stability, and swelling property of collagen-based scaffolds. Our ultimate goal is to further enhance the functional outcomes of MSC-based IVD regeneration by effectively solving the cell leakage problem. In this study, we aim to (1) fabricate a photochemically cross-linked collagen annulus plug; (2) evaluate the performance of the plug ex vivo by conducting mechanical push-out test with different loading protocols and cell leakage test; (3) design a clinically applicable delivery device for the plug and (4) evaluate the performance of the plug in vivo by using a rabbit IVD degeneration model. Materials and Methods Acid soluble rat tail type I collagen at 4 mg/mL was poured into cylindrical-shaped container and placed in an alkaline vapor chamber containing ammonium hydroxide to initiate the gelation process for 1 hour. The reconstituted collagen gel was equilibrated with rose Bengal solution (0.0005%) overnight before irradiating the gel Argon laser at 514 nm for 100 seconds at laser power of 0.2 W. The cross-linked collagen gel was then shaped into thin needles with length around 5 to 7 mm and diameter either less than 0.5 mm or around 1 mm by controlled dehydration. The plug was inserted into rabbit lumbar disk either through direct puncture or through a 21G-syringe needle. Histoacryl glue was used to seal the insertion site. The lumbar discs were then subjected to axial loading of 40–50N at 1 Hz for 7 days with either a protocol of continuous cyclic compression or a protocol simulating the physiological daily activities with cyclic and static loading patterns to evaluate the mechanical durability of the plug. In a separate experiment, quantum dots-labeled MSCs were injected into the disk and injection portal was immediately blocked with the annulus plug with and without Histoacryl glue. The discs were subjected to the physiological loading protocol and the culture medium was collected after 7 days to evaluate the cell leakage by visual inspection under a fluorescent microscopy. Results Gently pressing the plug-inserted disk immediately after insertion with fingers resulted in pushing out of the plug. This is because the dried plug is usually slightly smaller than the inner diameter of the needle and takes time to rehydrate to press-fit the injection portal. Sealing the injection site with Histoacryl glue prevents the ejection of plug immediately after insertion and allows rehydration of the plug before subjecting to physiological loading. Preliminary results on the push-out tests demonstrated that all collagen plugs, two using continuous cyclic compression and four using physiological loading patterns, were kept in their original position, that is, were not pushed out after 7 days of loading at a force comparable to the body weight of rabbits. Moreover, the culture medium collected from the quantum-dot labeled MSCs injection experiment showed no cell leakage after 7 days compression. Conclusion BioGlue is necessary to provide an initial sealing of the injection portal. The photochemically cross-linked collagen annulus plug are mechanically durable under ex vivo physiologically relevant loading but in vivo evaluation is necessary. The plug may be able to effectively block cell leakage but more ex vivo tests and in vivo evaluation are necessary. Finally, a custom-made special needle is being designed, fabricated and, evaluated for easy delivery of the plug in vivo. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared

Columns of calcium alginate gel pellets have excellent physical properties when used as a cell immobilization support. Columns of pellets were very resistant to compression and abrasion during passage of high concentrations of sucrose at high flow rates, but if the pellets were formed using low alginate and Ca2+ concentrations, compression occurred and flow out of the column was reduced and pressure built up. Transfer of sucrose into the pellets was controlled by internal diffusion, the rate of diffusion being increased by reductions in the alginate and Ca2+ concentrations used for immobilization and by the presence of entrapped active cells. Some leakage of cells occurred during use especially when cell division of the entrapped cells took place, but leakage could be minimized by using more highly polymerized pellets. Therefore, immobilization conditions can be chosen so as to form strong pellets, possessing high substrate transfer rates and low rates of cell leakage.

A new immobilization method which prevents cell leakage from the polymeric matrix into the medium was developed. In this method, the cells are entrapped in alginate fibers with double gel layers. The cells are restricted to the inner layer only while the outer layer helps to prevent cell leakage. When this system was tested for continuous fermentation, normal ethanol production was maintained for 300 h without cell leakage from the polymeric matrix.

Summary Addition of 100 μg/ml of Colistin (polymyxin E) to an actively-growing culture of Mycobacterium aurum caused apparent loss of viability starting after 20 min of drug addition, whereas significant Mg++ efflux was apparent within 5 min of drug addition. Therefore, Mg++ efflux preceded cell death by at least 15 min. These data confirmed earlier reports showing that Colistin interacted with the cytoplasmic membrane of mycobacteria.