Enzyme-Linked Immunofiltration Assay To Estimate Attachment of Thiobacilli to Pyrite

An enzyme-linked immunofiltration assay (ELIFA) has been developed in order to estimate directly and specifically Thiobacillus ferrooxidans attachment on sulfide minerals. This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for a subsequent immunoenzymatic reaction in microtiter plates. The polyclonal antiserum used in this study was raised against T. ferrooxidans DSM 583 and recognized cell surface antigens present on bacteria belonging to the genus Thiobacillus. This antiserum and the ELIFA allowed the direct quantification of attached bacteria with high sensitivity (10⁴ bacteria were detected per well of the microtiter plate). The mean value of bacterial attachment has been estimated to be about 10⁵ bacteria mg⁻¹ of pyrite at a particle size of 56 to 65 μm. The geometric coverage ratio of pyrite by T. ferrooxidans ranged from 0.25 to 2.25%. This suggests an attachment of T. ferrooxidans on the pyrite surface to well-defined limited sites with specific electrochemical or surface properties. ELIFA was shown to be compatible with the measurement of variable levels of adhesion. Therefore, this method may be used to establish adhesion isotherms of T. ferrooxidans on various sulfide minerals exhibiting different physicochemical properties in order to understand the mechanisms of bacterial interaction with mineral surfaces.     

Receptor‐mediated recognition of mycobacterial pathogens

Mycobacteria are a genus of bacteria that range from the non‐pathogenic Mycobacterium smegmatis to Mycobacterium tuberculosis, the causative agent of tuberculosis in humans. Mycobacteria primarily infect host tissues through inhalation or ingestion. They are phagocytosed by host macrophages and dendritic cells. Here, conserved pathogen‐associated molecular patterns (PAMPs) on the surface of mycobacteria are recognized by phagocytic pattern recognition receptors (PRRs). Several families of PRRs have been shown to non‐opsonically recognize mycobacterial PAMPs, including membrane‐bound C‐type lectin receptors, membrane‐bound and cytosolic Toll‐like receptors and cytosolic NOD‐like receptors. Recently, a possible role for intracellular cytosolic PRRs in the recognition of mycobacterial pathogens has been proposed. Here, we discuss currentideas on receptor‐mediated recognition of mycobacterial pathogens by macrophages and dendritic cells.     

IgM promotes the clearance of small particles and apoptotic microparticles by macrophages

Background

Antibodies are often involved in enhancing particle clearance by macrophages. Although the mechanisms of antibody-dependent phagocytosis have been studied for IgG in greater detail, very little is known about IgM-mediated clearance. It has been generally considered that IgM does not support phagocytosis. Recent studies indicate that natural IgM is important to clear microbes and other bioparticles, and that shape is critical to particle uptake by macrophages; however, the relevance of IgM and particle size in their clearance remains unclear. Here we show that IgM has a size-dependent effect on clearance.

Methodology/Principal Findings

We used antibody-opsonized sheep red blood cells, different size beads and apoptotic cells to determine the effect of human and mouse IgM on phagocytosis by mouse alveolar macrophages. Our microscopy (light, epifluorescence, confocal) and flow cytometry data show that IgM greatly enhances the clearance of small particles (about 1–2 micron) by these macrophages. There is an inverse relationship between IgM-mediated clearance by macrophages and the particle size; however, macrophages bind and internalize many different size particles coated with IgG. We also show that IgM avidly binds to small size late apoptotic cells or bodies (2–5 micron) and apoptotic microparticles (<2 µm) released from dying cells. IgM also promotes the binding and uptake of microparticle-coated beads.

Conclusions/Significance

Therefore, while the shape of the particles is important for non-opsonized particle uptake, the particle size matters for antibody-mediated clearance by macrophages. IgM particularly promotes the clearance of small size particles. This finding may have wider implications in IgM-mediated clearing of antigens, microbial pathogens and dying cells by the host.     

New Mechanisms to Explain the Effects of Added Lactose Fines on the Dispersion Performance of Adhesive Mixtures for Inhalation

Fine excipient particles or ‘fines’ have been shown to improve the dispersion performance of carrier-based formulations for dry powder inhalation. Mechanistic formulation studies have focussed mainly on explaining this positive effect. Previous studies have shown that higher drug contents may cause a decrease in dispersion performance, and there is no reason why this should not be true for fines with a similar shape, size and cohesiveness as drug particles. Therefore, the effects on drug detachment of ‘fine lactose fines’ (FLF, X₅₀ = 1.95 µm) with a similar size and shape as micronised budesonide were studied and compared to those of ‘coarse lactose fines’ (CLF, X₅₀ = 3.94 µm). Furthermore, interactions with the inhalation flow rate, the drug content and the mixing order were taken into account. The observed effects of FLF are comparable to drug content effects in that the detached drug fraction was decreased at low drug content and low flow rates but increased at higher flow rates. At high drug content the effects of added FLF were negligible. In contrast, CLF resulted in higher detached drug fractions at all flow rates and drug contents. The results from this study suggest that the effects of fines may be explained by two new mechanisms in addition to those previously proposed. Firstly, fines below a certain size may increase the effectiveness of press-on forces or cause the formation of strongly coherent fine particle networks on the carrier surface containing the drug particles. Secondly, when coarse enough, fines may prevent the formation of, or disrupt such fine particle networks, possibly through a lowering of their tensile strength. It is recommended that future mechanistic studies are based on the recognition that added fines may have any effect on dispersion performance, which is determined by the formulation and dispersion conditions.     

An Unaccounted Fraction of Marine Biogenic CaCO3 Particles

Biogenic production and sedimentation of calcium carbonate in the ocean, referred to as the carbonate pump, has profound implications for the ocean carbon cycle, and relate both to global climate, ocean acidification and the geological past. In marine pelagic environments coccolithophores, foraminifera and pteropods have been considered the main calcifying organisms. Here, we document the presence of an abundant, previously unaccounted fraction of marine calcium carbonate particles in seawater, presumably formed by bacteria or in relation to extracellular polymeric substances. The particles occur in a variety of different morphologies, in a size range from <1 to >100 µm, and in a typical concentration of 10⁴–10⁵ particles L⁻¹ (size range counted 1–100 µm). Quantitative estimates of annual averages suggests that the pure calcium particles we counted in the 1–100 µm size range account for 2–4 times more CaCO₃ than the dominating coccolithophoride Emiliania huxleyi and for 21% of the total concentration of particulate calcium. Due to their high density, we hypothesize that the particles sediment rapidly, and therefore contribute significantly to the export of carbon and alkalinity from surface waters. The biological and environmental factors affecting the formation of these particles and possible impact of this process on global atmospheric CO₂ remains to be investigated.     

Electrolyte Effects on Attachment of an Estuarine Bacterium

The effect of electrolyte concentration on attachment of Vibrio alginolyticus to hydroxyapatite was determined. Bacterial affinity for attachment to the surface and surface capacity were derived from linearization of bacterial adsorption isotherms. At low concentrations (<0.1 M) the affinity of the bacteria for the surface increased with increasing ionic strength, in agreement with the D.L.V.O. theory of colloid interaction. At higher concentrations, bacterial affinity for the surface decreased with increasing concentration of cations and was not related to ionic strength changes in the medium. These results demonstrate a change in the mechanism by which salts affect bacterial attachment at salt concentrations above 0.1 M. The results are consistent with the relationship between the proportion of attached bacteria and salinity observed in previously published field studies. The results may also resolve differences between various attachment studies carried out in different ionic strength media, utilizing different bacteria, surfaces, and experimental methods.     

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g. by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-Pérot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a ''real time'' observation of bacteria attachment.The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 10⁴ cells/ml) in less than an hour.     

A Novel Attempt to Standardize Results of CFD Simulations Basing on Spatial Configuration of Aortic Stent-Grafts

Currently, studies connected with Computational Fluid Dynamic (CFD) techniques focus on assessing hemodynamic of blood flow in vessels in different conditions e.g. after stent-graft’s placement. The paper propose a novel method of standardization of results obtained from calculations of stent-grafts'' “pushing forces” (cumulative WSS—Wall Shear Stress), and describes its usefulness in diagnostic process. AngioCT data from 27 patients were used to reconstruct 3D geometries of stent-grafts which next were used to create respective reference cylinders. We made an assumption that both the side surface and the height of a stent-graft and a reference cylinder were equal. The proposed algorithm in conjunction with a stent-graft “pushing forces” on an implant wall, allowed us to determine which spatial configuration of a stent-graft predispose to the higher risk of its migration. For stent-grafts close to cylindrical shape (shape factor φ close to 1) WSS value was about 267Pa, while for stent-grafts different from cylindrical shape (φ close to 2) WSS value was about 635Pa. It was also noticed that deformation in the stent-graft’s bifurcation part impaired blood flow hemodynamic. Concluding the proposed algorithm of standardization proved its usefulness in estimating the WSS values that may be useful in diagnostic process. Angular bends or tortuosity in bifurcations of an aortic implant should be considered in further studies of estimation of the risk of implantation failure.     

Analysis of the Murine Immune Response to Pulmonary Delivery of Precisely Fabricated Nano- and Microscale Particles

Nanomedicine has the potential to transform clinical care in the 21^st century. However, a precise understanding of how nanomaterial design parameters such as size, shape and composition affect the mammalian immune system is a prerequisite for the realization of nanomedicine''s translational promise. Herein, we make use of the recently developed Particle Replication in Non-wetting Template (PRINT) fabrication process to precisely fabricate particles across and the nano- and micro-scale with defined shapes and compositions to address the role of particle design parameters on the murine innate immune response in both in vitro and in vivo settings. We find that particles composed of either the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) or the biocompatible polymer polyethylene glycol (PEG) do not cause release of pro-inflammatory cytokines nor inflammasome activation in bone marrow-derived macrophages. When instilled into the lungs of mice, particle composition and size can augment the number and type of innate immune cells recruited to the lungs without triggering inflammatory responses as assayed by cytokine release and histopathology. Smaller particles (80×320 nm) are more readily taken up in vivo by monocytes and macrophages than larger particles (6 µm diameter), yet particles of all tested sizes remained in the lungs for up to 7 days without clearance or triggering of host immunity. These results suggest rational design of nanoparticle physical parameters can be used for sustained and localized delivery of therapeutics to the lungs.     

Aerobic Anoxygenic Phototrophic Bacteria Attached to Particles in Turbid Waters of the Delaware and Chesapeake Estuaries

Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that, if abundant, may be biogeochemically important in the oceans. We used epifluorescence microscopy and quantitative PCR (qPCR) to examine the abundance of these bacteria by enumerating cells with bacteriochlorophyll a (bChl a) and the light-reaction center gene pufM, respectively. In the surface waters of the Delaware estuary, AAP bacteria were abundant, comprising up to 34% of prokaryotes, although the percentage varied greatly with location and season. On average, AAP bacteria made up 12% of the community as measured by microscopy and 17% by qPCR. In the surface waters of the Chesapeake, AAP bacteria were less abundant, averaging 6% of prokaryotes. AAP bacterial abundance was significantly correlated with light attenuation (r = 0.50) and ammonium (r = 0.42) and nitrate (r = 0.71) concentrations. Often, bChl a-containing bacteria were mostly attached to particles (31 to 94% of total AAP bacteria), while usually 20% or less of total prokaryotes were associated with particles. Of the cells containing pufM, up to 87% were associated with particles, but the overall average of particle-attached cells was 15%. These data suggest that AAP bacteria are particularly competitive in these two estuaries, in part due to attachment to particles.     

Cellular,particle and environmental parameters influencing attachment in surface waters: a review

Effective modelling of the fate and transport of water‐borne pathogens is needed to support federally required pollution‐reduction plans, for water quality improvement planning, and to protect public health. Lack of understanding of microbial–particle interactions in water bodies has sometimes led to the assumption that bacteria move in surface waters not associated with suspended mineral and organic particles, despite a growing body of evidence suggesting otherwise. Limited information exists regarding the factors driving interactions between micro‐organisms and particles in surface waters. This review discusses cellular, particle and environmental factors potentially influencing interactions and in‐stream transport. Bacterial attachment in the aquatic environment can be influenced by properties of the cell such as genetic predisposition and physiological state, surface structures such as flagella and fimbriae, the hydrophobicity and electrostatic charge of the cell surface, and the presence of outer‐membrane proteins and extracellular polymeric substances. The mechanisms and degree of attachment are also affected by characteristics of mineral and organic particles including the size, surface area, charge and hydrophobicity. Environmental conditions such as the solution chemistry and temperature are also known to play an important role. Just as the size and surface of chemical particles can be highly variable, bacterial attachment mechanisms are also diverse.     

Effects of Inorganic Particles on Metabolism by a Periphytic Marine Bacterium

Measurements were made of adsorption of a periphytic marine bacterium, glucose, and glutamic acid to inorganic particles in seawater and defined bacterial growth medium. Measurements of the metabolism of bacteria were made in the presence and absence of particles by microcalorimetry and radiorespirometry. It was found that hydroxyapatite adsorbs glutamic acid, but not glucose, from the experimental medium. It was also found that hydroxyapatite adsorbs essentially all of the bacteria from the medium when the bacterial concentration is approximately 6 × 10⁵ bacteria per ml. If the bacterial concentration is approximately 6 × 10⁷, then only a small fraction of cells become attached. It was therefore possible to select bacterial concentrations and organic nutrients so that bacterial attachment, organic nutrient adsorption, or both would occur in different experiments. In this experimental system the metabolism by attached and nonattached bacteria of adsorbing and nonadsorbing organic nutrients was measured. The results show that bacterial activity in this model system was not enhanced by the particles, regardless of whether the bacteria, the organic nutrient, or both were associated with the surface. In fact, the respiratory activity of the attached bacteria was diminished in comparison with that of free bacteria.     

Multipurpose Love acoustic wave immunosensor for bacteria,virus or proteins detection

A multipurpose Love acoustic wave biosensor is described in this article. As mass loading is one of the main effect involved in acoustic wave sensors, a great range of biomolecules could be detected using such sensors. In this way, the antibody/antigen binding property was used to immobilise the target species. We first compared different coupling agents to link the antibodies sensitive layer to the SiO₂ sensor surface. Results showed that GPTS monolayer, allowing covalent attachment of antibodies bioreceptors, is better suited than DTSP and protein G. It permits to obtain a dense, stable and reproducible sensitive layer of antibodies. Then, different biological species with different size and shape like proteins, bacteriophages or bacteria were detected using such sensor. Different models have been chosen to validate the effective detection of a large species range: an anti-mouse antibody has been used to simulate small molecules (< 10 nm) like proteins or toxins, bacteriophage M13 for species lower than 1 μm like virus, and Escherichia coli for bacteria which are typically longer than 1 μm. Each kind of species were successfully quickly detected from few seconds for small proteins to one hour for bacteria, with detection threshold down to 4 ng/mm² for protein and 10⁶ cfu per milliliter for bacteria.     

Exopolysaccharide production and attachment strength of bacteria and diatoms on substrates with different surface tensions

Attachment strength and exopolysaccharide (EPS) production of Pseudomonas sp. (bacteria) and the diatom Amphora coffaeformis were studied on six different substrata with surface tensions between 19 and 64.5 mN m^–1. Test panels of the materials were exposed to bacterial cultures between 3 and 120 hours, and to diatom cultures between 48 and 72 hours. Exopolysaccharide production by surface-associated cells was measured using the phenol sulfuric acid method. Attachment studies were run by exposing test panels to laminar flow pressure using a radial flow chamber. Highest EPS production by bacteria and diatoms was recorded on substrata with surface tensions above 30 mN m^–1. Lowest EPS production occurred on substrata between 20 and 25 mN m^–1. Highest EPS production and strongest adhesion was found on polycarbonate (33.5 mN m^–1). Both test organisms improved their attachment strength with exposure time on most materials. However, amounts of produced EPS and improvement of attachment indicated that mechanisms other than polysaccharide production are more important on substrata with low surface tensions (<25 mN m^–1). Simply producing more polysaccharides is not sufficient to overcome weak attachment on materials with low surface tensions. For example, adhesion of Pseudomonas sp. and A. coffaeformis on polytetrafluorethylene/perfluor-copolymer (PFA; 22 mN m^–1). and glass (64.5 mN m^–1. was equally strong although EPS production was much higher on glass than on PFA. This is somewhat surprising for A. coffaeformis because polysaccharide production has been considered the most important attachment mechanism of A. coffaeformis.     

Explaining Bacterial Dispersion on Leaf Surfaces with an Individual-Based Model (PHYLLOSIM)

We developed the individual-based model PHYLLOSIM to explain observed variation in the size of bacterial clusters on plant leaf surfaces (the phyllosphere). Specifically, we tested how different ‘waterscapes’ impacted the diffusion of nutrients from the leaf interior to the surface and the growth of individual bacteria on these nutrients. In the ‘null’ model or more complex ‘patchy’ models, the surface was covered with a continuous water film or with water drops of equal or different volumes, respectively. While these models predicted the growth of individual bacterial immigrants into clusters of variable sizes, they were unable to reproduce experimentally derived, previously published patterns of dispersion which were characterized by a much larger variation in cluster sizes and a disproportionate occurrence of clusters consisting of only one or two bacteria. The fit of model predictions to experimental data was about equally poor (<5%) regardless of whether the water films were continuous or patchy. Only by allowing individual bacteria to detach from developing clusters and re-attach elsewhere to start a new cluster, did PHYLLOSIM come much closer to reproducing experimental observations. The goodness of fit including detachment increased to about 70–80% for all waterscapes. Predictions of this ‘detachment’ model were further supported by the visualization and quantification of bacterial detachment and attachment events at an agarose-water interface. Thus, both model and experiment suggest that detachment of bacterial cells from clusters is an important mechanism underlying bacterial exploration of the phyllosphere.     

Surfactant Protein D Binds to Coxiella burnetii and Results in a Decrease in Interactions with Murine Alveolar Macrophages

Coxiella burnetii is a Gram-negative, obligate intracellular bacterium and the causative agent of Q fever. Infections are usually acquired after inhalation of contaminated particles, where C. burnetii infects its cellular target cells, alveolar macrophages. Respiratory pathogens encounter the C-type lectin surfactant protein D (SP-D) during the course of natural infection. SP-D is a component of the innate immune response in the lungs and other mucosal surfaces. Many Gram-negative pulmonary pathogens interact with SP-D, which can cause aggregation, bactericidal effects and aid in bacterial clearance. Here we show that SP-D binds to C. burnetii in a calcium-dependent manner with no detectable bacterial aggregation or bactericidal effects. Since SP-D interactions with bacteria often alter macrophage interactions, it was determined that SP-D treatment resulted in a significant decrease in C. burnetii interactions to a mouse alveolar macrophage model cell line MH-S indicating SP-D causes a significant decrease in phagocytosis. The ability of SP-D to modulate macrophage activation by C. burnetii was tested and it was determined that SP-D does not alter the correlates measured for macrophage activation. Taken together these studies support those demonstrating limited activation of alveolar macrophages with C. burnetii and demonstrate interactions with SP-D participate in reduction of phagocyte attachment and phagocytosis.     

Impacts of Goethite Particles on UV Disinfection of Drinking Water

A unique association between bacterial cells and small goethite particles (~0.2 by 2 μm) protected Escherichia coli and Pseudomonas putida from UV inactivation. The protection increased with the particle concentration in the turbidity range of 1 to 50 nephelometric turbidity units and with the bacterium-particle attachment time prior to UV irradiation. The lower degree of bacterial inactivation at longer attachment time was mostly attributed to the particle aggregation surrounding bacteria that provided shielding from UV radiation.     

De-Novo Design of Antimicrobial Peptides for Plant Protection

This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of “healthy” food, these peptides might serve as templates for novel antibacterial and antifungal agents.