Biomedical subjects

Thomas R Neu

Publications and source records attributed to Thomas R Neu.

10 recordsLinked to original sources

PubMed2006

Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae.

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

Alginates↗

PubMed2006

Bacterial extracellular DNA forming a defined network-like structure.

It is generally assumed that nucleic acids are localized inside of living cells and that their primary function is the storage of information. In contrast, extracellular DNA is mainly considered as a remnant of lysed cells. Here, we report the formation of extracellular bacterial DNA as a spatial structure. An aquatic bacterium, strain F8, was isolated, which produced a stable filamentous network of extracellular DNA. Different staining and enzymatic techniques confirmed that it was DNA. We were able to amplify the 16S rRNA gene from the extracellular DNA. Restriction endonuclease cleavage and randomly amplified polymorphic DNA analysis of extracellular and genomic DNAs revealed major similarities, but also some differences in both sequences. Our data demonstrate a new function and relevance for extracellular DNA.

Bacteria↗

PubMed2005

Three stages of a biofilm community developing at the liquid-liquid interface between polychlorinated biphenyls and water.

Soil contaminated with polychlorinated biphenyls (PCB) was used as an inoculum to grow a complex biofilm community on PCB oil (Aroclor 1242) on a substratum (Permanox). The biofilm was monitored for 31 days by confocal laser scanning microscopy, community fingerprinting using single-strand conformational polymorphism (SSCP), amplicons of the 16S rRNA genes, and chemical analyses of the PCB congeners. SSCP analysis of the young biofilm revealed a rather diverse microbial community with species of the genera Herbaspirillum and Bradyrhizobium as dominant members. The biofilm developing on the PCB droplets displayed pronounced stages of PCB degradation and biofilm development not described before from pure-culture experiments. The first step was the colonization of the substratum while the PCB oil was hardly populated. When a certain density of bacteria was reached on the Permanox, the PCB was colonized, but soon the degradation of the congeners was markedly reduced and many cells were damaged, as seen by LIVE/DEAD staining. Finally, the biofilm formed aggregates and invaded the PCB oil, showing lower numbers of damaged cells than before and a dramatic increase in PCB degradation. This sequence of biofilm formation is understood as a maturation process prior to PCB oil colonization. This is followed by a thin biofilm on the PCB droplet, an aggregation process forming pockets in the PCB, and finally an invasion of the biofilm into the PCB oil. Only the mature biofilm showed degradation of pentachlorinated PCB congeners, which may be reductively dechlorinated and the resulting trichlorobiphenyls then aerobically metabolized.

Bacteria↗

PubMed2005

Effects of selected pharmaceuticals on riverine biofilm communities.

Although pharmaceutical and therapeutic products are widely found in the natural environment, there is limited understanding of their ecological effects. Here we used rotating annular bioreactors to assess the impact of 10 microg.L(-1) of the selected pharmaceuticals ibuprofen, carbamazepine, furosemide, and caffeine on riverine biofilms. After 8 weeks of development, community structure was assessed using in situ microscopic analyses, fluor-conjugated lectin binding, standard plate counts, fluorescent in situ hybridization, carbon utilization spectra, and stable carbon isotope analyses. The biofilm communities varied markedly in architecture although only caffeine treated biofilms were significantly thicker. Cyanobacteria were suppressed by all 4 compounds, whereas the nitrogen containing caffeine, furosemide, and carbamazepine increased algal biomass. Ibuprofen and carbamazepine reduced bacterial biomass, while caffeine and furosemide increased it. Exopolymer content and composition of the biofilms was also influenced. Significant positive and negative effects were observed in carbon utilization spectra. In situ hybridization analyses indicated all treatments significantly decreased the gamma-proteobacterial populations and increased beta-proteobacteria. Ibuprofen in particular increased the alpha-proteobacteria, beta-proteobacteria, cytophaga-flavobacteria, and SRB385 probe positive populations. Caffeine and carbamazepine additions resulted in significant increases in the high GC354c and low GC69a probe positive cells. Live-dead analyses of the biofilms indicated that all treatments influenced the ratio of live-to-dead cells with controls having a ratio of 2.4, carbamazepine and ibuprofen being 3.2 and 3.5, respectively, and furosemide and caffeine being 1.9 and 1.7, respectively. Stable isotope analyses of the biofilms indicated delta 13C values shifted to more negative values relative to control biofilms. This shift may be consistent with proportional loss of cyanobacteria and relative increase in algal biomass rather than incorporation of pharmaceutical carbon into microbial biofilm. Thus, at 10 microg.L(-1) levels pharmaceuticals exhibit both nutrient-like and toxic effects on riverine microbial communities.

Bacteria↗

PubMed2004

Community structure and photosynthetic activity of epilithon from a highly acidic (pH < or = 2) mountain stream in Patagonia, Argentina.

We explored a benthic community living on stones in an acidic (pH< or =2) stream of active volcanic origin from Patagonia, Argentina, by combining in situ measurements (temperature, pH, conductivity, dissolved oxygen), photosynthesis of intact biofilms (measured with microsensors by the light-dark shift method), pure-culture experiments on isolated algae, and confocal laser scanning microscopy on the biofilms. The epilithon of the Agrio River was dominated (99% of total biomass) by one species: Gloeochrysis (Chrysophyceae). This species was observed as brown, mucilaginous, 200-microm-thick films on stones, growing in clumps in a dense matrix of fungal hyphae, bacteria, and inorganic particles held together by extracellular polymeric substances. Gloeochrysis was isolated and cultivated. The photosynthetic rate measured at saturation irradiance was 120 micromol oxygen (mg chlorophyll a)(-1) h(-1) under laboratory conditions, and the saturation rate of photosynthesis by carbon dioxide was 90 micromol oxygen (mg chlorophyll a)(-1) h(-1) for oxygen evolution. Photosynthetic activity of the biofilm was light-dependent and saturated above 200 micromol photons m(-2) s(-1). In the dark, the stone surface became anoxic. Our data suggest that primary production in the Agrio River was not limited by light, carbon, or phosphorus but instead, nitrogen-limited.

Argentina↗

PubMed2004

Three-dimensional differentiation of photo-autotrophic biofilm constituents by multi-channel laser scanning microscopy (single-photon and two-photon excitation).

A simple microscopic method to three-dimensionally differentiate between various members in photo-autotrophic biofilm systems is described. By dual-channel single-photon (confocal) and two-photon laser scanning microscopy, the signals in the red and far red channels as well as their combination can be simultaneously recorded. The method takes advantage of the autofluorescent signal of cyanobacteria-recorded in the red and far red channel and the autofluorescent signal of the green algae-recorded in the far red channel only. The differentiation is based on the specific pigment composition of cyanobacteria and green algae in combination with the appropriate filter settings for detection of the autofluorescent emission signals. The method allows the non-destructive, three-dimensional examination of fully hydrated interfacial microbial communities at high resolution as well as the clear separation between autofluorescent signals of cyanobacteria and green algae. Furthermore, there is a third option to record additional signals simultaneously such as nucleic acid stained bacteria, bacteria labeled with phylogenetic probes or glycoconjugates stained by using lectins. With state of the art laser scanning microscopes, even a fourth channel is available for recording yet another parameter, e.g. in the reflection (single-photon only) or fluorescence (single- and two-photon) mode. Thus the approach represents a convenient tool to study multiple parameters of complex photo-autotrophic biofilm systems.

Biofilms↗

PubMed2003

Photolysis and biodegradation of selected resin acids in River Saale water, Germany.

The River Saale is the Elbe's major tributary flowing through the state of Thuringia, Germany and receives organics inputs from several industrial facilities including pulp and paper mills. Resin acids constitute a major class of polar organics and environmental toxins derived primarily from pulp and paper processing of softwoods. Since wastewater treatment methods at pulp and paper mills are not always capable of removing the persistent resin acids prior to effluent discharge, alternative or complementary degradation methods may be required. Here, the facile photodegradation of four resin acids--abietic, dehydroabietic, isopimaric, and pimaric--was observed with pseudo-first-order kinetics when exposed to broad band and UV254-radiation. Further experimentation in rotating annular biofilm reactors with UV-exposed and unexposed River Saale water spiked with abietic and dehydroabietic acids indicated that photolysis is an effective pretreatment method for resin acid biodegradation. The bacterial toxicity of the aqueous resin acids solutions as measured with Microtox luminescence assays decreased with exposure time. Consequently, photo- and biodegradation of the resin acids did not generate any notable amounts of toxic intermediates and/or the intermediates formed were further degraded into compounds of lower toxicity than the parents. With tandem photo- and biological treatment at pulp and paper mills, as well as in-situ degradation by solar radiation and natural biofilms within the River Saale, resin acid inputs can be reduced in both concentration and toxicity to near undetectable levels with little or no ecological significance.

Acids↗

PubMed2002

Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding-analysis.

A technique combining fluorescent in situ hybridization and lectin-binding-analysis (FISH-LBA) was developed and applied for the simultaneous detection of cellular components and glycoconjugates in lotic microbial aggregates (river snow). River snow aggregates were directly collected from the bulk water phase into coverslip chambers, in which the complete procedure including fixation, fluorescent in situ hybridization, lectin-binding and optical analysis by confocal laser scanning microscopy was performed. Neither autofluorescence originating from phyotosynthetic organisms nor inorganic particles did negatively interfere with the FISH-LBA technique. In river snow samples obtained from the river Elbe, Germany, distinct compartments of the river snow structure could be visualized with FITC-labelled lectins from Triticum vulgaris, Limulus polyphemus, Arachis hypogaea, Phaseolus vulgaris and Pseudomonas aeruginosa, binding to frequently occurring saccharide residues in the river snow matrix. The analysis could be performed on different levels of complexity. The combined technique visualized bacteria of different phylogenetic groups in the entire river snow structure as well as glycoconjugate components linked with various microcolonies. Different lectins stained slime layers and cell-envelopes of individual eukaryotic and prokaryotic cells. Consequently, application of the FISH-LBA technique allows the linkage between cellular and glycoconjugate identity in complex microbial communities.

Animals↗

PubMed2002

Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms.

A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F(420)-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.

Biofilms↗

PubMed2001

Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems.

An assessment of lectin-binding analysis for the characterization of extracellular glycoconjugates as part of the extracellular polymeric substances in environmental microbial communities was performed using fully hydrated river biofilms. The applicability of the method was evaluated for single, dual and triple staining with a panel of fluor-conjugated lectins. It was shown that lectin-binding analysis was able to stain glycoconjugates within biofilm communities. Lectin staining also demonstrated spatial heterogeneity within the biofilm matrix. Furthermore, the application of two or even three lectins was possible if suitable combinations were selected. The lectin-binding analysis can be combined with general nucleic acid stains to collect both nucleic acid and glycoconjugate signals. The effects of incubation time, lectin concentration, fluor labelling, carbohydrate inhibition, order of addition and lectin interactions were studied. An incubation time of 20 min was found to be sufficient for completion of lectin binding. It was not possible to ascertain saturating concentration for individual lectins, therefore a standard concentration was used for the assay. Carbohydrate inhibition tests indicated that fluorescein isothiocyanate (FITC)-conjugated lectins had more specific binding characteristics than tetramethyl rhodamine isothiocyanate (TRITC)- or cyanine dye (CY5)-labelled lectins. The order of addition and the nature of the fluor conjugate were also found to influence the binding pattern of the lectins. Therefore the selection of a panel of lectins for investigating the EPS matrix must be based on a full evaluation of their behaviour in the biofilm system to be studied. Despite this necessity, lectin-binding analysis represents a valuable tool to examine the glycoconjugate distribution in fully hydrated biofilms. Thereby, chemical heterogeneities within extracellular biofilm locations can be identified in order to examine the role (e.g. sorption properties, microenvironments, cell-extracellular polymeric substance interactions) of the extracellular polymeric substances in environmental biofilm systems.

Bacteria↗