Lars Fledelius Rickelt

Polymorphonuclear Leukocytes Restrict Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients

ABSTRACT Cystic fibrosis (CF) patients have increased susceptibility to chronic lung infections by Pseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate the in vivo growth physiology of P. aeruginosa within lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescence in situ hybridization (PNA-FISH)-based method was used to estimate the in vivo growth rates of P. aeruginosa directly in lung tissue samples from CF patients and the growth rates of P. aeruginosa in infected lungs in a mouse model. The growth rate of P. aeruginosa within CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect on P. aeruginosa by PMNs was also observed in vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding that P. aeruginosa growth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O2 consumption, which slows the growth of P. aeruginosa in infected CF lungs. In support of this, the growth of P. aeruginosa was significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronic P. aeruginosa infection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.

Combined Imaging of Bacteria and Oxygen in Biofilms

ABSTRACT Transparent sensors for microscopic O2 imaging were developed by spin coating an ultrathin (<1- to 2-μm) layer of a luminescent O2 indicator onto coverslips. The sensors showed (i) an ideal Stern-Volmer quenching behavior of the luminescence lifetime towards O2 levels, (ii) homogeneous measuring characteristics over the sensor surface, and (iii) a linear decline of luminescence lifetime with increasing temperature. When a batch of such coverslip sensors has been characterized, their use is thus essentially calibration free at a known temperature. The sensors are easy to use in flow chambers and other growth devices used in microbiology. We present the first application for combined imaging of O2 and bacteria in a biofilm flow chamber mounted on a microscope equipped with a spinning-disk confocal unit and a luminescence lifetime camera system.

Ultrabright planar optodes for luminescence life-time based microscopic imaging of O2 dynamics in biofilms

New transparent optodes for life-time based microscopic imaging of O₂ were developed by spin-coating a μm-thin layer of a highly luminescent cyclometalated iridium(III) coumarin complex in polystyrene onto glass cover slips. Compared to similar thin-film O₂ optodes based on a ruthenium(II) polypyridyl complex or a platinum(II) porphyrin, the new planar sensors have i) higher brightness allowing for much shorter exposure times and thus higher time resolution, ii) more homogeneous and smaller pixel to pixel variation over the sensor area resulting in less noisy O₂ images, and iii) a lower temperature dependency simplifying calibration procedures. We used the new optodes for microscopic imaging of the spatio-temporal O₂ dynamics at the base of heterotrophic biofilms in combination with confocal imaging of bacterial biomass and biofilm structure. This allowed us to directly link biomass distribution to O₂ distribution under both steady state and non-steady state conditions. We demonstrate that the O₂ dynamics in biofilms is governed by a complex interaction between biomass distribution, mass transfer and flow that cannot be directly inferred from structural information on biomass distribution alone.

A simple optode based method for imaging O2 distribution and dynamics in tap water biofilms

A ratiometric luminescence intensity imaging approach is presented, which enables spatial O2 measurements in biofilm reactors with transparent planar O2 optodes. Optodes consist of an O2 sensitive luminescent dye immobilized in a 1-10 μm thick polymeric layer on a transparent carrier, e.g. a glass window. The method is based on sequential imaging of the O2 dependent luminescence intensity, which are subsequently normalized with luminescent intensity images recorded under anoxic conditions. We present 2-dimensional O2 distribution images at the base of a tap water biofilm measured with the new ratiometric method and compare the results with O2 distribution images obtained in the same biofilm reactor with luminescence lifetime imaging. Using conventional digital cameras, such simple normalized luminescence intensity imaging can yield images of 2-dimensional O2 distributions with a high signal-to-noise ratio and spatial resolution comparable or even surpassing those obtained with expensive and complex luminescence lifetime imaging systems. The method can be applied to biofilm growth incubators allowing intermittent experimental shifts to anoxic conditions or in systems, in which the O2 concentration is depleted during incubation.

An Optode Sensor Array for Long-Term In Situ Oxygen Measurements in Soil and Sediment

Long-term measurements of molecular oxygen (O) dynamics in wetlands are highly relevant for understanding the effects of water level changes on net greenhouse gas budgets in these ecosystems. However, such measurements have been limited due to a lack of suitable measuring equipment. We constructed an O optode sensor array for long-term in situ measurements in soil and sediment. The new device consists of a 1.3-m-long, cylindrical, spear-shaped rod equipped with 10 sensor spots along the shaft. Each spot contains a thermocouple fixed with a robust fiberoptic O optode made by immobilizing a layer of Pt(II) meso-tetra(pentafluorophenyl)porphine in polystyrene at the end of a 2-mm polymethyl methacrylate plastic fiber. Temperature and O optode readings are collected continuously by a data logger and a multichannel fiberoptic O meter. The construction and measuring characteristics of the sensor array system are presented along with a novel approach for temperature compensation of O optodes. During in situ application over several months in a peat bog, we used the new device to document pronounced variations in O distribution after marked shifts in water level. The measurements showed anoxic conditions below the water level but also diel variations in O concentrations in the upper layer presumably due to rhizospheric oxidation by the main vegetation The new field instrument thus enables new and more detailed insights to the in situ O dynamics in wetlands.

Fiber-Optic Probes for Small-Scale Measurements of Scalar Irradiance

A new method for producing fiber‐optic microprobes for scalar irradiance (=fluence rate) measurements is described. Such fine‐scale measurements are important in many photobiological disciplines. With the new method, it is possible to cast spherical 30–600 μm wide light integrating sensor tips onto tapered or untapered optical fibers. The sensor tip is constructed by first casting a clear polymethyl methacrylate (PMMA) sphere (~80% of the size of the final probe tip diameter) onto the optical fiber via dip‐coating. Subsequently, the clear sphere is covered with light diffusing layers of PMMA mixed with TiO2 until the fiber probe exhibits a satisfactory isotropic response (typically ±5–10%). We also present an experimental setup for measuring the isotropic response of fiber‐optic scalar irradiance probes in air and water. The fiber probes can be mounted in a syringe equipped with a needle, facilitating retraction of the spherical fiber tip. This makes it, e.g. possible to cut a hole in cohesive tissue with the needle before inserting the probe. The light‐collecting properties of differently sized scalar irradiance probes (30, 40, 100, 300 and 470 μm) produced by this new method were compared to probes produced with previously published methods. The new scalar irradiance probes showed both higher throughput of light, especially for blue light, as well as a better isotropic light collection over a wide spectral range. The new method also allowed manufacturing of significantly smaller scalar irradiance microprobes (down to 30 μm tip diameter) than hitherto possible, and such sensors allow minimally invasive high‐resolution scalar irradiance measurements in thin biofilms, leaves and animal tissues.

Tachykinins Stimulate Release of Peptide Hormones (Glucagon-Like Peptide-1) and Paracrine (Somatostatin) and Neurotransmitter (Vasoactive Intestinal Polypeptide) from Porcine Ileum Through NK-1 Receptors

The effects of infusion of the two tachykinins,substance P (SP) and neurokinin A (NKA), and ofcapsaicin on the release of glucagon-like peptide-1(GLP-1), somatostatin, and vasoactive intestinalpolypeptide (VIP) were studied in isolated, vascularlyperfused ileal segments. SP (10-8 M) stimulated GLP-1,somatostatin, and VIP release to 141.8 ± 6.6% (N= 18), 230.3 ± 38.7% (N = 21), and 359.7 ±60.5% (N = 22) of basal output, respectively. NKA(10-8 M) only stimulated VIP release (to181.2 ± 16.7% of basal release, N = 22). Theeffects of SP and NKA were blocked by the NK-1 receptorantagonist CP96345 (10-6 M). Infusion of atropine(10-6 M) had no effect on the SP-inducedGLP-1 release, but partly inhibited the effect of SP onsomatostatin and VIP release, and the effect of NKA onVIP release. Capsaicin infusions (10-5 M) significantlystimulated both GLP-1, somatostatin, and VIP release to111.1 ± 4.5% (N = 9), 138.0 ± 15.8% (N =9) and 208.3 ± 63.8% (N = 8) of basal release,respectively. Simultaneous addition of receptor antagonists to all threetachykinin receptors (CP96345, SR48968, and SR142801,all at 10-6 M) significantly inhibited theeffect of capsaicin on VIP release, whereas the releaseof GLP-1 and somatostatin was unaffected. Weconclude that tachykinins potently stimulate the releaseof GLP-1, somatostatin, and VIP in the porcine ileum viaNK-1 receptors. The effect on somatostatin and VIP is partly mediated via cholinergic neurons.Sensory neurons releasing tachykinins could be involvedin the regulation of VIPergic neurons.