Process analysis of biofilms by photoacoustic spectroscopy

Biofilms are aggregates of microorganisms and biopolymers which occur at aqueous interfaces. Biofilms play an important role in the degradation of pollutants in natural water systems as well as in wastewater treatment plants. In this communication, the use of photoacoustic spectroscopy (PAS) as a new biofilm monitoring technique is presented. PAS combines features of optical spectroscopy and ultrasonic tomography and allows a depth-resolved analysis of optically and acoustically inhomogeneous media. For the first time, both biofilm and bulk liquid were monitored by photoacoustic sensor heads. In this way, sorption of suspended iron(III) oxide particles on the outer and inner surfaces of the biofilm could be observed on-line and in situ. Colloids can act as carriers of pollutants and influence stability and degradation efficiency of biofilms.     

Dynamic removal of oral biofilms by bubbles

A novel approach to the removal of biofilms from solid surfaces is to pass large numbers of air bubbles over the surfaces. Such a phenomenon occurs when teeth are brushed with some types of powered toothbrushes that accelerate bubbly fluid against or across teeth surfaces. Video recordings of air bubbles propelled against a mature biofilm of Streptococcus mutans showed that the bubbles removed the biofilm at the point of collision. A mathematical model of the removal process was proposed and was able to simulate the kinetics of the biofilm removal process. Removal rate was modeled to be proportional to the bubble footprint area and the number of collisions per time. The fraction of biofilm removed per bubble collision is on the order of 0.4, a value much larger than would have been expected based on previous research employing bubbles that moved slowly along a surface that was partially covered with adherent bacteria. The higher removal efficiency is attributed to fluid dynamic shear forces that occur in conjunction with the thermodynamic forces that pull bacteria from a surface as a bubble contacts the biofilm. Fast bubbly flow is expected to remove bacterial biofilm from hard surfaces such as teeth.     

Disruption of supported lipid bilayers by semihydrophobic nanoparticles

Understanding the interaction between functional nanoparticles and cell membranes is critical to use nanomaterials for broad biomedical applications with minimal cytotoxicity. In this work, we have investigated the effect of adsorbed semihydrophobic nanoparticles (NPs) on the dynamics and morphology of model cell membranes. We have systematically varied the degree of surface hydrophobicity of carboxyl end-functionalized polystyrene NPs of varied size in buffer solutions with varied ionic strength. It is observed that semihydrophobic NPs can readily adsorb on neutral SLBs and drag lipids from SLBs to NP surfaces. Above a critical NP concentration, the disruption of SLBs is observed, accompanied with the formation and rapid growth of lipid-poor regions on NP-adsorbed SLBs. In the study of the effect of solution ionic strength on NP surface hydrophobic degree and the growth of lipid-poor regions, we have concluded that the hydrophobic interaction enhanced by screened electrostatic interaction underlies the envelopment of NPs by lipids that are attracted from SLBs to the surface of NPs or their aggregates. Hence, the formation and growth of lipid-poor regions, or vaguely referred as "pores" or "holes" in the literature, can be controlled by NP concentration, size, and surface hydrophobicity, which is critical to design functional nanomaterials for effective nanomedicine while minimizing possible cytotoxicity.     

Superoxide dismutase activity in some strains of lactobacilli: induction by manganese

Dialyzed cell-free extract of lactobacilli was found to contain superoxide dismutase activity by using a test system in which superoxide ion is generated by xanthine oxidase. The specific activities of Lactobacillus acidophilus ATCC 4356, Lactobacillus murinus ATCC 35020, Lactobacillus acidophilus CRL 358, Lactobacillus plantarum ATCC 8014, Lactobacillus casei CRL 431, Lactobacillus plantarum CRL 353, Lactobacillus fermentum ATCC 9338, Lactobacillus buchneri NCDO 110, and Lactobacillus fermentum CRL 251 were between 0.06 and 0.43 U/mg protein. The presence of superoxide dismutase activity was demonstrated when the strains were grown in media containing Mn2+ ions. Superoxide dismutase of lactobacilli may be an Mn enzyme since it was not inhibited by either cyanide or azide ions. However, the cell-free extract of Lactobacillus murinus ATCC 35020 contains superoxide dismutase activity sensitive to both ions.     

Disruption of DNA repair processes by carcinogenic metal compounds

Based on pronounced enhancing effects in combination with other DNA-damaging agents the potentials of Ni(II), Cd(II) and As(III) to interfere with DNA repair processes in HeLa cells was investigated. With respect to oxidative DNA damage, Ni(II) and Cd(II) induced DNA strand breaks starting at concentrations of 250 μM and 5 μM, respectively. The induction of oxidative DNA base modifications like 8-hydroxyguanine was restricted to the cytotoxic concentration of 750 μM Ni(II) and not observed after treatment with Cd(II). In contrast, the removal of oxidative DNA base modifications was inhibited at concentrations as low as 50 μM Ni(II) and 0.5 μM Cd(II). Regarding nucleotide excision repair, Ni(II) and Cd(II) disturbed the DNA-protein interactions involved in the damage recognition step when applying HeLa nuclear protein extracts and a UV-damaged oligonucleotide, while As(III) inhibited the actual incision event. In the case of Ni(II) and Cd(II), this effect was reversible by the addition of Mg(II) and Zn(II), respectively. Furthermore, Cd(II) inactivated the isolated bacterial Fpg protein, most likely by the displacement of Zn(II) from its zinc finger structure. Since DNA is continuously damaged by exogenous and endogenous sources, an impaired repair capacity might well account for the carcinogenic action of the metal compounds. Received: 30 July 1997 / Revised: 6 October 1997 / Accepted: 10 October 1997     

Uranium speciation in biofilms studied by laser fluorescence techniques

Biofilms may immobilize toxic heavy metals in the environment and thereby influence their migration behaviour. The mechanisms of these processes are currently not understood, because the complexity of such biofilms creates many discrete geochemical microenvironments which may differ from the surrounding bulk solution in their bacterial diversity, their prevailing geochemical properties, e.g. pH and dissolved oxygen concentration, the presence of organic molecules, e.g. metabolites, and many more, all of which may affect metal speciation. To obtain such information, which is necessary for performance assessment studies or the development of new cost-effective strategies for cleaning waste waters, it is very important to develop new non-invasive methods applicable to study the interactions of metals within biofilm systems. Laser fluorescence techniques have some superior features, above all very high sensitivity for fluorescent heavy metals. An approach combining confocal laser scanning microscopy and laser-induced fluorescence spectroscopy for study of the interactions of biofilms with uranium is presented. It was found that coupling these techniques furnishes a promising tool for in-situ non-invasive study of fluorescent heavy metals within biofilm systems. Information on uranium speciation and uranium redox states can be obtained.     

Molecular interactions in biofilms

A biofilm may be defined as a microbially derived, sessile community characterized by cells that attach to an interface, embed in a matrix of exopolysaccharide, and demonstrate an altered phenotype. This review covers the current understanding of the nature of biofilms and the impact that molecular interactions may have on biofilm development and phenotype using the motile gram-negative rod Pseudomonas aeruginosa and the nonmotile gram-positive cocci Staphylococcus aureus as examples.     

GC-MS检测萃取剂中内分泌干扰物

烷基酚作为内分泌干扰物的代表物成为环境毒理学研究的热点,多支链取代也最难生物降解.在湿法冶金工业中壬基酚作为基质改善剂在大量使用[1],而且萃取剂主要品种多含壬基酚结构单元,它们通过降解将释放出壬基酚,为此建立对壬基酚的分析检测十分有必要,尤其是西部开发更应注意新工艺后边有许多新的化学药品还未进行的环境评价,应防止类似农药666的错误再次出现.萃取剂中壬基酚的GC-MS检测方法以国内外3种主产品为对象,其分析样品编号分别为12102,12103,12104,壬基酚对照编号为12101.样品直接溶于甲醇中进样.     

A new approach to non-destructive analysis of biofilms by confocal Raman microscopy

Confocal Raman microscopy (CRM) of biofilms enables one to determine the distribution of different microorganisms and other substances inside physiological intact microbial communities. These biofilms are of outstanding interest for biological wastewater treatment. In contrast to invasive techniques, such as fluorescent in situ hybridization (FISH), we were able to identify anaerobically ammonium-oxidising (anammox) bacteria without pretreatment processes of the samples just by its Raman vibrational signature. The presented results provide new insights into the complex interactions of different organisms in microbial communities without interfering with them.     

Inhibition and dispersion of proteobacterial biofilms

A small molecule derived from a marine natural product with the ability to inhibit biofilm formation and also disperse established proteobacterial biofilms is presented.     

Ring-Modified Histidine-Containing Cationic Short Peptides Exhibit Anticryptococcal Activity by Cellular Disruption

Delineation of clinical complications secondary to fungal infections, such as cryptococcal meningitis, and the concurrent emergence of multidrug resistance in large population subsets necessitates the need for the development of new classes of antifungals. Herein, we report a series of ring-modified histidine-containing short cationic peptides exhibiting anticryptococcal activity via membrane lysis. The N-1 position of histidine was benzylated, followed by iodination at the C-5 position via electrophilic iodination, and the dipeptides were obtained after coupling with tryptophan. In vitro analysis revealed that peptides Trp-His[1-(3,5-di-tert-butylbenzyl)-5-iodo]-OMe (10d, IC₅₀ = 2.20 μg/mL; MIC = 4.01 μg/mL) and Trp-His[1-(2-iodophenyl)-5-iodo)]-OMe (10o, IC₅₀ = 2.52 μg/mL; MIC = 4.59 μg/mL) exhibit promising antifungal activities against C. neoformans. When administered in combination with standard drug amphotericin B (Amp B), a significant synergism was observed, with 4- to 16-fold increase in the potencies of both peptides and Amp B. Electron microscopy analysis with SEM and TEM showed that the dipeptides primarily act via membrane disruption, leading to pore formation and causing cell lysis. After entering the cells, the peptides interact with the intracellular components as demonstrated by confocal laser scanning microscopy (CLSM).     

Functional artificial free-standing yeast biofilms

Here we report fabrication of artificial free-standing yeast biofilms built using sacrificial calcium carbonate-coated templates and layer-by-layer assembly of extracellular matrix-mimicking polyelectrolyte multilayers. The free-standing biofilms are freely floating multilayered films of oppositely charged polyelectrolytes and live cells incorporated in the polyelectrolyte layers. Such biofilms were initially formed on glass substrates of circular and ribbon-like shapes coated with thin layers of calcium carbonate microparticles. The templates were then coated with cationic and anionic polyelectrolytes to produce a supporting multilayered thin film. Then the yeast alone or mixed with various micro- and nanoparticle inclusions was deposited onto the multilayer composite films and further coated with outer polyelectrolyte multilayers. To detach the biofilms from the glass substrates the calcium carbonate layer was chemically dissolved yielding free-standing composite biofilms. These artificial biofilms to a certain degree mimic the primitive multicellular and colonial species. We have demonstrated the added functionality of the free-standing artificial biofilms containing magnetic, latex and silver micro- and nanoparticles. We have also developed "symbiotic" multicellular biofilms containing yeast and bacteria. This approach for fabrication of free-standing artificial biofilms can be potentially helpful in development of artificial colonial microorganisms composed of several different unicellular species and an important tool for growing cell cultures free of supporting substrates.     

Small molecule control of bacterial biofilms

Bacterial biofilms are defined as a surface attached community of bacteria embedded in a matrix of extracellular polymeric substances that they have produced. When in the biofilm state, bacteria are more resistant to antibiotics and the host immune response than are their planktonic counterparts. Biofilms are increasingly recognized as being significant in human disease, accounting for 80% of bacterial infections in the body and diseases associated with bacterial biofilms include: lung infections of cystic fibrosis patients, colitis, urethritis, conjunctivitis, otitis, endocarditis and periodontitis. Additionally, biofilm infections of indwelling medical devices are of particular concern, as once the device is colonized infection is virtually impossible to eradicate. Given the prominence of biofilms in infectious diseases, there has been an increased effort toward the development of small molecules that will modulate bacterial biofilm development and maintenance. In this review, we highlight the development of small molecules that inhibit and/or disperse bacterial biofilms through non-microbicidal mechanisms. The review discuses the numerous approaches that have been applied to the discovery of lead small molecules that mediate biofilm development. These approaches are grouped into: (1) the identification and development of small molecules that target one of the bacterial signaling pathways involved in biofilm regulation, (2) chemical library screening for compounds with anti-biofilm activity, and (3) the identification of natural products that possess anti-biofilm activity, and the chemical manipulation of these natural products to obtain analogues with increased activity.     

Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was applied successfully to characterize the penetration and diffusion capabilities of fluorescent probes (latex beads and fluorescein isothiocyanate-dextran) of different size and electrical charge in two models of monomicrobial biofilms with low (Lactococcus lactis biofilm) or high (Stenotrophonas maltophilia biofilm) contents of extracellular polymeric substance (EPS). FCS measurements performed on each biofilm can show deviation from Brownian diffusion, depending on the local structure of the biofilm and the fluorophore size. In this case, we fitted the data to an anomalous diffusion model and determined apparent diffusion coefficients, which can be 50 times smaller than the values in aqueous solutions. This result was interpreted as steric hindrance of the diffusion of the fluorescent particles within the biofilm that can lead to a total inhibition as observed particularly in the mushroom-like structure of the S. maltophilia biofilm. Alternatively, mechanisms for the absence of FCS signal behavior were related to attractive electrostatic interactions between cationic particles and negatively charged bacteria or to specific interactions between dextrans and EPS of the biofilm matrix.     

Disruption of HepG2 cell adhesion by gold nanoparticle and Paclitaxel disclosed by in situ QCM measurement

Cell adhesion is a crucial issue for cytotoxicity or anticancer effectiveness for tumor cells. However, how both nanoparticles and drugs affect cell adhesion has not yet been defined. Herein, we report for the first time that gold nanoparticles and Paclitaxel can disrupt adhesion, as well as enhance apoptosis of HepG2 cell individually and synergistically, as observed by in situ measurement using quartz crystal microbalance (QCM). It was also found by MTT assay that gold nanoparticles of low cellular cytotoxicity enhance the antiproliferation and apoptosis of HepG2 cell induced by Paclitaxel. Those findings would be of great potential for biomedical application of nanoparticles.