Comparison of the cytotoxic effect of polystyrene latex nanoparticles on planktonic cells and bacterial biofilms

The cytotoxic effect of positively charged polystyrene latex nanoparticles (PSL NPs) was compared between planktonic bacterial cells and bacterial biofilms using confocal laser scanning microscopy, atomic force microscopy, and a colony counting method. Pseudomonas fluorescens, which is commonly used in biofilm studies, was employed as the model bacteria. We found that the negatively charged bacterial surface of the planktonic cells was almost completely covered with positively charged PSL NPs, leading to cell death, as indicated by the NP concentration being greater than that required to achieve single layer coverage. In addition, the relationship between surface coverage and cell viability of P. fluorescens cells correlated well with the findings in other bacterial cells (Escherichia coli and Lactococcus lactis). However, most of the bacterial cells that formed the biofilm were viable despite the positively charged PSL NPs being highly toxic to planktonic bacterial cells. This indicated that bacterial cells embedded in the biofilm were protected by self-produced extracellular polymeric substances (EPS) that provide resistance to antibacterial agents. In conclusion, mature biofilms covered with EPS exhibit resistance to NP toxicity as well as antibacterial agents.     

Mutual information theory for biomedical applications: Estimation of three protein-adsorbed dialysis membranes

Protein-adsorbed dialysis membranes are evaluated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical imaging technique. Protein adsorption causing permeability change is one of big issues in the development of dialysis membranes. Bovine serum albumin adsorption onto three kinds of dialysis membranes has been evaluated with TOF-SIMS. In the present study three kinds of proteins, bovine serum albumin, α-chymotripsinogen A, and cytochrome C adsorbed onto hollow-fiber dialysis membranes, were measured by means of TOF-SIMS and then TOF-SIMS spectra were analyzed using mutual information. Then specific peaks of fragment ions related to α-chymotripsinogen A and bovine serum albumin were found, respectively. In this condition, however, specific peaks to cytochrome C were not able to find compared with other samples. Finally, chemical images of α-chymotripsinogen A and bovine serum albumin, respectively, adsorbed onto the membranes with co-existing proteins were obtained. The results of TOF-SIMS images of the proteins on the membranes show different tendency of adsorption depending on co-existing proteins. Further study is needed to study more detailed protein adsorption onto the membranes with co-existing proteins.     

Inhibition of methicillin resistant Staphylococcus aureus by a plasma needle

In numerous recent papers plasma chemistry of non equilibrium plasma sources operating at atmospheric pressure has been linked to plasma medical effects including sterilization. In this paper we present a study of the effectiveness of an atmospheric pressure plasma source, known as plasma needle, in inhibition of the growth of biofilm produced by methicillin resistant Staphylococcus aureus (MRSA). Even at the lowest powers the biofilms formed by inoculi of MRSA of 10⁴ and 10⁵ CFU have been strongly affected by plasma and growth in biofilms was inhibited. The eradication of the already formed biofilm was not achieved and it is required to go to more effective sources.     

In situ TEM and SEM studies on the antimicrobial activity and prevention of Candida albicans biofilm by Cassia spectabilis extract

The inhibitory effect of Cassia spectabilis methanol leaf extract was evaluated against biofilm forming Candida albicans, which was sensitive to 6.25 mg/ml concentration of the extract. Transmission (TEM) and scanning electron microscope (SEM) observations were used to study the anticandidal activity and prevention of biofilm formation by the C. spectabilis extract. SEM analysis further revealed reduction in C. albicans biofilm in response to the extract. The main abnormalities noted via TEM study was the alterations in morphology and complete collapse of the yeast cells after 36 h of exposure to the extract. The significant antifungal activity shown by this methanol extract of C. spectabilis suggests its potential against infections caused by C. albicans.     

Leucine pools in Escherichia coli biofilm discovered by Raman imaging

In structured communities of bacteria known as biofilms, a variety of biomolecules have been shown to play a unique role as signals and/or regulators in biofilm formation. Here, we report that high levels of the amino acid leucine (leucine pool) were detected, for the first time, within microcolonies in a 30‐h‐old Escherichia coli biofilm by Raman imaging. Localization of leucine revealed by multifrequency Raman images indicates leucine accumulation during the early stage of the E. coli biofilm formation, which may have resulted from physiological environment‐specific metabolic adaptation. We demonstrate that our label‐free Raman imaging method provides a useful platform for directly identifying still unknown natural products produced in biofilms as well as for visualizing heterogeneous distributions of biofilm constituents in situ. Copyright © 2011 John Wiley & Sons, Ltd.     

Prevention of biofilm re‐development on Ti‐6Al‐4V alloy by cometary discharge with a metallic grid

The influence of a non‐thermal plasma (NTP) on the gram‐negative bacteria Escherichia coli and Pseudomonas aeruginosa, the gram‐positive bacterium Staphylococcus epidermidis, and the yeast Candida albicans grown on agar or in the biofilm form was compared. NTP was produced by a DC cometary discharge. The biofilms were grown on the surface of Ti‐6Al‐4V alloy often used in the manufacture of prosthetic implants. The exposure by NTP not only inhibited the surface growth of microorganisms in agar cultures but also significantly suppressed the viability of bacteria and yeast in biofilms and prevented its re‐developed from persistent cells remaining in the lower layers of the biofilm. An almost complete prevention of biofilm re‐development was achieved in the case of S. epidermidis; other microorganisms displayed substantial lowering of biofilm biomass and its metabolic activity.     

The Inhibition Effect of Graphene Oxide Nanosheets on the Development of Streptococcus mutans Biofilms

Bacterial biofilms play a critical role in dental pathogenic mechanisms due to their ability to tolerate many traditional antibacterial agents. Thus, it is crucial to find effective methods to inhibit the occurrence and development of biofilms. Recently, graphene oxide (GO) nanosheets have become a promising antibacterial nanomaterial for use in various applications. This study focuses on the effect of GO nanosheets on the development of Streptococcus mutans biofilms. It is found that GO nanosheets are highly effective at inhibiting the formation of bacterial biofilms at concentrations ranging from low to high in early development stages. After GO treatment, the ratio of living cells in the biofilm decreases significantly, and the biofilm structure is destroyed. In contrast, GO has a minimal effect on mature biofilms.     

Investigation of the cosmetic ingredient distribution in the stratum corneum using NanoSIMS imaging

In order to understand the mechanisms of action of cosmetic ingredients, it is important to establish the distribution of the component agents within the epidermis of the skin. To date, time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to detect cosmetic ingredients in the skin. However, it is technically difficult to investigate the distribution of the agents in the stratum corneum using TOF-SIMS. Therefore, an analytical method with higher spatial resolution is required. In this study, we investigated an imaging analysis technique based on NanoSIMS to detect cosmetic ingredients in the skin. Pig skin was used as a model for human skin. The sample was treated with a cosmetic formulation containing ¹⁵N-labelled pseudo-ceramide (SLE). The sample was frozen with liquid nitrogen and cross-sections were cut using a cryomicrotome. As a result, the fine layer structure of the corneocytes was clearly observed by using NanoSIMS. Our studies indicate that SLE penetrates into the stratum corneum via an intercellular route. We conclude that application of NanoSIMS analysis can contribute to a better understanding of the function of cosmetic ingredients in the skin.     

Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor

Microorganisms that colonize surfaces, biofilms, are of significant importance due to their role in medical infections, subsurface contaminant remediation, and industrial processing. Spatially resolved data on the distribution of biomass within a capillary bioreactor, the heterogeneity of the biofilm itself and the impact on transport dynamics for a Staphylococcus epidermidis biofilm in the natural growth state are presented. The data demonstrate the ability of magnetic resonance microscopy to study spatially resolved processes in bacterial biofilms, thus providing a basis for future studies of spatially resolved metabolism and in vivo clinical detection.     

How does ultrasonic cavitation remove dental bacterial biofilm?

Bacterial biofilm accumulation is problematic in many areas, leading to biofouling in the marine environment and the food industry, and infections in healthcare. Physical disruption of biofilms has become an important area of research. In dentistry, biofilm removal is essential to maintain health. The aim of this study is to observe biofilm disruption due to cavitation generated by a dental ultrasonic scaler (P5XS, Acteon) using a high speed camera and determine how this is achieved. Streptococcus sanguinis biofilm was grown on Thermanox™ coverslips (Nunc, USA) for 4 days. After fixing and staining with crystal violet, biofilm removal was imaged using a high speed camera (AX200, Photron). An ultrasonic scaler tip (tip 10P) was held 2 mm away from the biofilm and operated for 2 s. Bubble oscillations were observed from high speed image sequences and image analysis was used to track bubble motion and calculate changes in bubble radius and velocity on the surface. The results demonstrate that most of the biofilm disruption occurs through cavitation bubbles contacting the surface within 2 s, whether individually or in cavitation clouds. Cleaning occurs through shape oscillating microbubbles on the surface as well as through fluid flow.     

TOF-SIMS analysis of magnetic materials in chum salmon head

A piece of tissue extracted from a chum salmon Oncorhynchus keta head was measured with time-of-flight secondary ion mass spectrometry (TOF-SIMS) in order to evaluate the distribution and composition of magnetic materials in the tissue, which may concern with geomagnetic navigation of long-distance migrating salmon. Several depositions of iron compounds were detected in the tissue by TOF-SIMS analysis. Comparing with total ion images providing a topological tissue structure, specific distribution of iron ion in the tissue was clearly shown. Higher magnification TOF-SIMS analysis revealed the existence of the aggregations of iron particles. Iron oxide clusters comprising many submicron particles were also detected in the tissue using scanning electron microscopy and X-ray analysis, suggesting the common existence of submicron-scale iron oxides in salmon heads. These results suggest that TOF-SIMS analysis is a valid method to clarify detailed structures and chemical properties of candidate magnetoreceptors in fish heads.     

Detection of algae and bacterial biofilms formed on titanium surfaces using micro-Raman analysis

Biofouling is one of the major impediments in the use of titanium in sea-water cooled condensers of power plants, which is otherwise an excellent material with respect to corrosion resistance. Raman microscopic experiments were carried out on biofilms grown on titanium surfaces to find out the chemical composition of complex extracellular polymeric substances (EPS) in the biofilm. Though the spectral resolution of normal Raman experiments on these systems was very poor, it was improved when micro-SERS experiments were carried out using mono and bimetallic Ag and Cu colloids. It was observed that spatial distribution of polysaccharides was higher than that of proteins in algae biofilms formed on titanium matrix. Similar experiments were performed on laboratory cultured bacterial films of Pseudomonas aeruginosa. It was evidenced that algal and bacterial biofilms on titanium can be clearly distinguished with the help of Raman mapping coupled with SERS technique using bimetallic Ag/Cu colloids.     

TOF-SIMS structural characterization of self-assembly monolayer of cytochrome b5 onto gold substrate

Orientation and three-dimensional structure of immobilized proteins on bio-devices are very important to assure their high performance. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is able to analyze upper surface of one layer of molecules. Orientation of immobilized proteins can be evaluated based on determination of a partial structure, representing ensemble of amino acids, on the surface part. In this study, a monolayer of cytochrome b5 was reconstituted onto gold substrate and investigated by surface plasmon resonance (SPR). After freeze-drying, the resulted protein self-assembly was evaluated using TOF-SIMS with the bismuth cluster ion source, and then TOF-SIMS spectra were analyzed to select peaks specific to cytochrome b5 and identify their chemical formula and ensembles of amino acids. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the modified cytochrome b5 and three-dimensional structure of cytochrome b5 registered in the protein data bank. Finally, fragment-ion-generating parts of the immobilized-cytochrome b5 are determined based on the suggested residues and three-dimensional structure. These results suggest the actual structure and confirm the expected orientation of immobilized protein.     

Fiber-optic sensors for the estimation of oxygen gradients within biofilms on metals

This paper presents an optical approach to measure the oxygen gradients in biofilms grown on stainless-steel 316L. The sensor is constructed by immobilizing an oxygen-quenchable fluorophore at the tip of an optical fiber. The response time and reversibility of fiber-optic sensor have been analyzed. By mounting the optical fiber using a computerized micromanipulator, the vertical profiles of oxygen are measured with 10 μm depth resolution across biofilms on metal coupons. Steep concentration gradients of oxygen have been observed within the biofilm, both perpendicular and parallel to the metal surface. Optical response of the biofilm grown metal surface is found to be a consistent measure of the oxygen profiles within biofilms.     

Ultrasonic irradiation enhanced the efficacy of antimicrobial photodynamic therapy against methicillin-resistant Staphylococcus aureus biofilm

Antimicrobial photodynamic therapy (aPDT) is a non-pharmacological antimicrobial regimen based on light, photosensitizer and oxygen. It has become a potential method to inactivate multidrug-resistant bacteria. However, limited by the delivery of photosensitizer (PS) in biofilm, eradicating biofilm-associated infections by aPDT remains challenging. This study aimed to explore the feasibility of combining ultrasonic irradiation with aPDT to enhance the efficacy of aPDT against methicillin-resistant staphylococcus aureus (MRSA) biofilm. A cationic benzylidene cyclopentanone photosensitizer with much higher selectivity to bacterial cells than mammalian cells were applied at the concentration of 10 μM. 532 nm laser (40 mW/cm², 10 min) and 1 MHz ultrasound (500 mW/cm², 10 min, simultaneously with aPDT) were employed against MRSA biofilms in vitro. In addition to combined with ultrasonic irradiation and aPDT, MRSA biofilms were treated with laser irradiation only, photosensitizer only, ultrasonic irradiation only, ultrasonic irradiation and photosensitizer, and aPDT respectively. The antibacterial efficacy was determined by XTT assay, and the penetration depth of PS in biofilm was observed using a photoluminescence spectrometer and a confocal laser scanning microscopy (CLSM). In addition, the viability of human dermal fibroblasts (WS-1 cells) after the same treatments mentioned above and the uptake of P3 by WS-1 cells after ultrasonic irradiation were detected by CCK-8 and CLSM in vitro. Results showed that the percent decrease in metabolic activity resulting from the US + aPDT group (75.76%) was higher than the sum of the aPDT group (44.14%) and the US group (9.88%), suggesting synergistic effects. Meanwhile, the diffusion of PS in the biofilm of MRSA was significantly increased by 1 MHz ultrasonic irradiation. Ultrasonic irradiation neither induced the PS uptake by WS-1 cells nor reduced the viability of WS-1 cells. These results suggested that 1 MHz ultrasonic irradiation significantly enhanced the efficacy of aPDT against MRSA biofilm by increasing the penetration depth of PS. In addition, the antibacterial efficacy of aPDT can be enhanced by ultrasonic irradiation, the US + aPDT treatment demonstrated encouraging in vivo antibacterial efficacy (1.73 log₁₀ CFU/mL reduction). In conclusion, the combination of aPDT and 1 MHz ultrasound is a potential and promising strategy to eradicate biofilm-associated infections of MRSA.     

The combination of ultrasound and chlorogenic acid to inactivate Staphylococcus aureus under planktonic,biofilm, and food systems

This study aimed to investigate the mechanism of different treatments, namely, ultrasound (US), chlorogenic acid (CA), and ultrasound combined with chlorogenic acid (US plus CA) on the inactivation of Staphylococcus aureus planktonic and biofilm cells. Results showed that the combined treatment of US and CA exhibited remarkable synergistic antibacterial and antibiofilm effects. Scanning electron microscopy images indicated that the combined treatment of US and CA caused the most serious damage to the cell morphology. Confocal laser scanning microscopy images revealed that the combined treatment led to sharp increase and severe damage to the permeability of the cell membrane, causing the release of ATP and nucleic acids and decreasing the exopolysaccharide contents in S. aureus biofilm. Finally, the combined treatment of US plus 1% CA for 60 min inactivated S. aureus cells by 1.13 lg CFU/g on mutton. Thus, the combined treatment of US and CA had synergistic effect against S. aureus under planktonic, biofilm, and food systems.     

Comparison of detection efficiencies of negatively charged gold-alkanethiolate-, gold-sulfur- and gold-clusters in ToF-SIMS

In order to improve quantification of high mass ions, the influence of cluster composition on detection efficiencies has been studied using a TOF-SIMS IV with the extended capability of postaccelerating ions up to 20 keV. In this experimental study, we focus on the comparison of detection efficiencies for three types of negatively charged secondary cluster ions: gold-alkanethiolate-clusters (Au_xM_y), gold-sulfur-clusters (Au_xS_y) and gold-clusters (Au_x). The clusters were sputtered from self-assembled monolayers of hexadecanethiols on gold substrates using 10 keV Ar⁺ primary ions. The detection efficiencies were derived on the basis of a function for the secondary electron yield and a fourth-order approximated Poisson probability distribution for electron propagation and amplification within the microchannel plate.In addition to the well-known dependence of detection efficiencies on ion mass and energy, which has already been studied for positively charged ions, we were able to show a similar behaviour for the investigated negatively charged secondary ions. We have observed major variations among the three types of clusters at similar mass and energy as predicted in a theoretical approach. The observed differences are due to the different composition of the investigated clusters which has a major influence on the kinetic ion induced electron emission within the microchannel plate. For the first time it was possible to experimentally verify these predictions for detection efficiencies.     

Light-induced antifungal activity of TiO2 nanoparticles/ZnO nanowires

Antifungal activity of TiO₂/ZnO nanostructures under visible light irradiation was investigated. A simple chemical method was used to synthesize ZnO nanowires. Zinc acetate dihydrate, Polyvinyl Pyrrolidone and deionized water were used as precursor, capping and solvent, respectively. TiO₂ nanoparticles were deposited on ZnO nanowires using an atmospheric pressure chemical vapor deposition system. X-ray diffraction pattern of TiO₂/ZnO nano-composite has represented the diffraction peaks relating to the crystal planes of the TiO₂ (anatase and rutile) and ZnO. TiO₂/ZnO nanostructure antifungal effect on Candida albicans biofilms was studied and compared with the activity of TiO₂ nanoparticles and ZnO nanowires. The high efficiency photocatalytic activity of TiO₂ nanoparticles leads to increased antifungal activity of ZnO nanowires. Scanning electron microscope was utilized to study the morphology of the as prepared nanostructures and the degradation of the yeast.     

Embedded structure of silicon monoxide in SiO2 films

The structure of SiO_x (x = 1.94) films has been investigated using both X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The SiO_x films were deposited by vacuum evaporation. XPS spectra show that SiO_1.94 films are composed of silicon suboxides and the SiO₂ matrix. Silicon clusters appeared only negligibly in the films in the XPS spectra. Si₃O⁺ ion species were found in the TOF-SIMS spectra with strong intensity. These results reveal the structure of the films to be silicon monoxide embedded in SiO₂, and this structure most likely exists as a predominant form of Si₃O₄. The existence of Si-Si structures in the SiO₂ matrix will give rise to dense parts in loose glass networks.