Legionella-like slender rods multiplying within a strain of Acanthamoeba sp. isolated from drinking water.
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Acanthamoeba↗
Biomedical subjects
R Amann
Publications and source records attributed to R Amann.
Secretoneurin, a novel neuropeptide, in the human dental pulp.
Secretoneurin is a neuropeptide that is stored in and released from primary afferent neurones. By radioimmunoassay and immunohistochemistry, secretoneurin was here demonstrated in the human dental pulp and localized in varicose nerve fibres that were frequently associated with blood vessels. No significant correlation was found between the levels of immunoreactive secretoneurin and immunoreactive calcitonin-gene related peptide. The results suggest that primary afferent neurones of the human dental pulp contain secretoneurin, which may influence local inflammatory responses if it is released together with other neuropeptides.
Adolescent↗
Application of 23S rDNA-targeted oligonucleotide probes specific for enterococci to water hygiene control.
Identification of enterococci species by hybridization with recently designed species-specific and group-specific 23S rDNA-targeted oligonucleotide probes was superior to results obtained with a common biochemical test panel. Considering these findings, a molecular biological procedure for the detection of enterococci in water samples was developed. A short enrichment is followed by an amplification step and a hybridization reaction in microtiter plate format. The detection limit is about 1 CFU/ml, and results are available within 26 h.
Bacterial Typing Techniques↗
Analysis of broad-scale differences in microbial community composition of two pristine forest soils.
Broad-scale differences in soil microbial community composition were analyzed in two contrasting soils using DNA reassociation and % G + C profiles for analysis on the community-level, and filter- and whole cell hybridization techniques for a coarse-level characterization of larger phylogenetic groups of bacteria. Reassociation analysis of DNA from bacterial fractions extracted from the organic soil Seim and the mineral soil Hau revealed similar complexity of the communities with 5700 and 4900 different bacterial genomes (g soil [dry wt])-1, respectively. Thermal denaturation studies showed wide % G + C distributions in DNA from bacteria of both soils. Differences in the median % G + C with 55 to 61% for the bacterial community in soil Seim and 61 to 66% for that in soil Hau indicated a higher proportion of bacteria with a high DNA G + C content in soil Hau. In situ hybridization with fluorescent (Cy3-labeled) probes targeting larger phylogenetic groups showed minor differences between both soils, and between direct detection of bacteria in dispersed soil slurries and in bacterial fractions extracted from soils through about 90% of the total bacteria were lost during extraction. In dispersed slurries of both soils, only probes ALF1b, SRB385, and PLA46 hybridized to cells accounting for more than 1% of the DAPI-stained cells, while numbers obtained after hybridization with probes ARCH915, BET42a, GAM42a, HGC69a, and CF319a were below the detection limit set at < 1%. These results were confirmed by in situ hybridization with horseradish peroxidase (HRP)-labeled probes and subsequent Cy3-tyramide signal amplification. In contrast, dot blot hybridization with probe HGC69a indicated significant amounts of Gram-positive bacteria with a high DNA G + C content in both soils. These could subsequently be visualized in non-dispersed soil slurries by in situ hybridization with HRP-labeled probe HGC69a and Cy3-tyramide signal amplification. Filamentous Gram-positive bacteria with a high DNA G + C content, likely actinomycetes, which are present in soil Hau in significant numbers are obviously destroyed by procedures used for soil dispersion.
Actinomycetales↗
In situ methods for assessment of microorganisms and their activities.
Recent technical developments in the field of molecular biology and microsensors are beginning to enable microbiologists to study the abundance, localization and activity of microorganisms in situ. The various new methods on their own bear high potential but it is the combination of studies on structure and function of microbial communities that will yield the most detailed insights in the way microorganisms operate in nature.
Bacteria↗
The nonpeptide B2 receptor antagonist FR173657: inhibition of effects of bradykinin related to its role in nociception.
1. The nonpeptide bradykinin B2 receptor antagonist, FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-(2, 4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl] phenyl]-N-methylaminocarbonylmethyl] acrylamide), was tested in models involving bradykinin-induced activation of primary afferent neurones in vitro and in vivo. 2. Bradykinin-induced contractions of the rabbit isolated iris sphincter muscle mediated by tachykinin release from trigeminal afferent neurones were inhibited in a non-competitive manner by FR173657. A pKB value of 7.9 was calculated. Effects of substance P were unaffected by FR173657. 3. Nociceptive behavioural responses following intraplantar injection of bradykinin in unanaesthetized rats were reduced by 0.3 micromol kg(-1) FR173657 s.c. (P < 0.05), and completely abolished by 3 micromol kg(-1) (P < 0.05). Peroral administration of 5 micromol kg(-1) FR173657 abolished the bradykinin effects (P < 0.05); lower doses had no significant effect. 4. Shortening by intraplantar injection of bradykinin of the paw withdrawal latency in response to radiant heat was abolished by 3 micromol kg(-1) FR173657 s.c. (P < 0.05), while 300 nmol kg(-1) had an intermediate effect. Hyperalgesia induced by prostaglandin E2 remained unaffected by FR173657. 5. Blood pressure reflexes following i.p. instillation of bradykinin in anaesthetized rats were inhibited by FR173657 s.c. with an ID50 of 1.1 micromol kg(-1), while the peptidic B2 antagonist icatibant (Hoe-140; D-Arg0-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin) caused inhibition at significantly lower doses (ID50 8.5 nmol kg(-1) P < 0.001). Responses to hydrochloric acid i.p. remained unaffected by FR173657. 6. FR173657 or similar nonpeptide compounds may be useful for the development of drugs for diseases involving pain induced by the release of endogenous kinins, i.e. especially in acute inflammatory conditions.
Analgesics↗
Bradykinin-evoked sensitization of neuropeptide release from afferent neurons in the guinea-pig lung.
1. It has been shown that bradykinin (BK) causes sensitization of airway sensory neurons and an enhancement of the cough reflex in guinea-pigs. In the present study, the guinea-pig isolated perfused lung was used to investigate the possible enhancement by BK of histamine-evoked neuropeptide release from peripheral terminals of primary afferent neurons, and to determine the contribution of cyclooxygenase products of arachidonate metabolism to this effect. 2. The lung was perfused with oxygenated physiological salt solution containing peptidase inhibitors (thiorphan, bestatin and captopril, 1 microM each). BK and histamine were added to the perfusate for 10 and 5 min, respectively. 3. BK alone (0.1 microM) evoked the release of 10.35+/-2.4 fmol immunoreactive calcitonin gene-related peptide (CGRP), histamine alone (100 microM) evoked the release of 12.7+/-1.6 fmol CGRP. Stimulation with 100 microM histamine in the presence of 0.1 microM BK (added 5 min before histamine and present during histamine) evoked the release of 67.1+/-5.3 fmol CGRP. 4. Prostaglandin (PG) release was stimulated by BK (418+/-71 pmol 15-keto-13,14-dihydro-PGF2alpha and 345+/-59 pmol 6-keto-PGF1alpha), and, to a lesser extent, by histamine (36.1+/-7.4 pmol 15-keto-13,14-dihydro-PGF2alpha, and 24.6+/-3.9 pmol 6-keto-PGF1alpha). Prostaglandin release induced by histamine in the presence of BK was not significantly higher than with BK alone. 5. Indomethacin (5 microM) as well as the bradykinin B2 receptor antagonist HOE140 (icatibant, 1 microM) inhibited prostaglandin release following stimulation with histamine in combination with BK. CGRP release evoked by histamine in combination with BK was attenuated by indomethacin and HOE140 to 22.1+/-7.8 fmol and 16.4+/-3.8 fmol, respectively, significantly less than the value obtained in control experiments (67.1+/-5.3 fmol). 6. The results suggest that BK-induced stimulation of prostaglandin synthesis results in facilitation of histamine-evoked release of pro-inflammatory neuropeptides from afferent neurons, a mechanism that probably becomes relevant during inflammation, and that can be blocked by a bradykinin B2 receptor antagonist.
Adrenergic beta-Antagonists↗
Seasonal community and population dynamics of pelagic bacteria and archaea in a high mountain lake
The seasonal variations in community structure and cell morphology of pelagic procaryotes from a high mountain lake (Gossenkollesee, Austria) were studied by in situ hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and image-analyzed microscopy. Compositional changes and biomass fluctuations within the assemblage were observed both in summer and beneath the winter ice cover and are discussed in the context of physicochemical and biotic parameters. Proteobacteria of the beta subclass (beta-proteobacteria) formed a dominant fraction of the bacterioplankton (annual mean, 24% of the total counts), whereas alpha-proteobacteria were of similar relative importance only during spring (mean, 11%). Bacteria of the Cytophaga-Flavobacterium cluster, although less abundant, constituted the largest fraction of the filamentous morphotypes during most of the year, thus contributing significantly to the total microbial biomass. Successive peaks of threadlike and rod-shaped archaea were observed during autumn thermal mixing and the period of ice cover formation, respectively. A set of oligonucleotide probes targeted to single phylotypes was constructed from 16S rRNA-encoding gene clone sequences. Three distinct populations of uncultivated microbes, affiliated with the alpha- and beta-proteobacteria, were subsequently monitored by FISH. About one-quarter of all of the beta-proteobacteria (range, 6 to 53%) could be assigned to only two phylotypes. The bacterial populations studied were annually recurrent, seasonally variable, and vertically stratified, except during the periods of lake overturn. Their variability clearly exceeded the fluctuations of the total microbial assemblage, suggesting that the apparent stability of total bacterioplankton abundances may mask highly dynamic community fluctuations.
Journal Article↗
Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes.
In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083(T). Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI; 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness with Eco1482, respectively) was as follows: I, 4%; II, 14%; III, 21%; IV, 29%, V, 19%; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of E. coli should facilitate a more rational selection of probe target sites for other species as well.
Base Sequence↗
Bacterial community dynamics during start-up of a trickle-bed bioreactor degrading aromatic compounds.
This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a care painting facility as the inoculum and Solvesso100 as the sole carbon source. The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides. Two significant shifts in the bacterial community structure could be demonstrated. The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria. A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor. The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined. Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor. In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis. Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.
Bacteria↗
Phylogeny and identification in situ of Nevskia ramosa.
An enrichment of the neuston bacterium Nevskia ramosa was investigated by the cultivation-independent rRNA approach. N. ramosa was first described by Famintzin in 1892 as a rod-shaped, slightly bent bacterium forming typical flat rosettes on the surface of shallow freshwater habitats by unilateral slime formation. PCR in combination with cloning and sequencing was used for retrieving 21 partial and 5 nearly full-length 16S rRNA sequences forming three tight clusters. In situ hybridization with rRNA-targeted oligonucleotide probes allowed us to assign the three sequence clusters to three distinct bacterial populations abundant in the enrichment. The two probes that unambiguously identified the N. ramosa morphotype were derived from a 16S rRNA sequence that had similarities of 87.9 to 88.9% to the rRNA sequences of the most closely related group in the database, Xanthomonas sp. and relatives. N. ramosa currently is the only representative of an independent, deep branch of the gamma subclass of the class Proteobacteria. The two other populations abundant in the enrichment were affiliated with the alpha subclass of the class Proteobacteria. They were most closely related to Blastobacter sp. (97.2% similarity) and Mycoplana bullata (97.6% similarity) and might represent new species in the respective genera.
Bacteria↗
Identification and activities in situ of Nitrosospira and Nitrospira spp. as dominant populations in a nitrifying fluidized bed reactor.
Bacterial aggregates from a chemolithoautotrophic, nitrifying fluidized bed reactor were investigated with microsensors and rRNA-based molecular techniques. The microprofiles of O2, NH4+, NO2-, and NO3- demonstrated the occurrence of complete nitrification in the outer 125 microgram of the aggregates. The ammonia oxidizers were identified as members of the Nitrosospira group by fluorescence in situ hybridization (FISH). No ammonia- or nitrite-oxidizing bacteria of the genus Nitrosomonas or Nitrobacter, respectively, could be detected by FISH. To identify the nitrite oxidizers, a 16S ribosomal DNA clone library was constructed and screened by denaturing gradient gel electrophoresis and selected clones were sequenced. The organisms represented by these sequences formed two phylogenetically distinct clusters affiliated with the nitrite oxidizer Nitrospira moscoviensis. 16S rRNA-targeted oligonucleotide probes were designed for in situ detection of these organisms. FISH analysis showed that the dominant populations of Nitrospira spp. and Nitrosospira spp. formed separate, dense clusters which were in contact with each other and occurred throughout the aggregate. A second, smaller, morphologically and genetically different population of Nitrospira spp. was restricted to the outer nitrifying zones.
Ammonia↗
Determination of nociceptin-like immunoreactivity in the rat dorsal spinal cord.
An endogenous ligand for the opioid receptor-like (ORL1) receptor has been characterized recently. This 17 amino acid peptide, nociceptin, has potent effects on pain processing in experimental animals. In the present study, we describe the development of a radio-immunoassay for nociceptin, suitable for quantification of immunoreactivity in the rat spinal cord. in the dorsal spinal cord we found 641 fmol nociceptin-like immunoreactivity (IR)/mg protein, while in the ventral spinal cord the amount was below the detection limit of the assay (11 fmol/mg protein). High pressure liquid chromatography (HPLC) showed that the immunoreactive material was heterogeneous with the major part eluting at the position of synthetic nociceptin. The presence of nociceptin in the dorsal spinal cord is compatible with its proposed involvement in modulating afferent transmission.
Animals↗
Release of calcitonin gene-related peptide in cardiac anaphylaxis.
We have investigated the antigen-stimulated release of calcitonin gene-related peptide (CGRP) from ovalbumin-sensitized guinea-pig isolated hearts and the interaction with other mediators of anaphylaxis released concomitantly. It was found that antigen challenge caused a significant increase of CGRP release (from basal 31.2 +/- 2.9 to 51.6 +/- 4.9 fmol/5 min). Anaphylactic CGRP release was significantly attenuated in the presence of the cyclooxygenase inhibitor indomethacin while the 5-lipoxygenase inhibitor Bay-X1005 ((R)-2-[4-quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) had no significant effect. Combined treatment with the histamine receptor (H1,H2) antagonists mepyramine and cimetidine also significantly attenuated anaphylactic release of CGRP. Under control conditions antigen injection increased release of cysteinyl-leukotrienes (LT), thromboxane (TXB2) and 6-keto-prostaglandin (PG)F1 alpha from basal values of 0.96 +/- 0.09, 2.7 +/- 0.7 and 3.4 +/- 0.28 ng/5 min respectively, to 5.9 +/- 0.9, 48.4 +/- 3.4 and 6.9 +/- 1.4 ng/5 min. Indomethacin abolished the release of cyclooxygenase products of arachidonate metabolism and simultaneously increased cysteinyl-LT release significantly (8.8 +/- 1.4 ng/5 min). Conversely Bay-X1005 completely abolished cysteinyl-LT release and had no significant effect on anaphylactic release of TXB2 and 6-keto-PGF1 alpha. Simultaneous blockade of H1 and H2 receptors abolished release of 6-keto-PGF1 alpha, while release of TXB2 and cysteinyl-LT was not significantly affected. The results indicate that CGRP is not a primary mediator of the immediate hypersensitivity reaction of the heart, but is in turn released by arachidonic acid metabolites of the cyclooxygenase pathway and histamine. In contrast, LT obviously do not contribute to anaphylactic CGRP release. CGRP is a potent coronary vasodilator and could act as endogenous functional antagonist of vasoconstrictor mediators also released during cardiac anaphylaxis such as cysteinyl-LT, platelet activating factor and TXA2.
6-Ketoprostaglandin F1 alpha↗
Effects of local anesthesia on substance P and CGRP content of the human dental pulp.
The aim of the study was to determine immunoreactive Substance P (iSP) and immunoreactive calcitonin gene-related peptide (iCGRP) content in the human dental pulp and whether local anesthesia has an effect on the neuropeptide content. Dental pulps were obtained from patients, who underwent surgical extraction of all 4 impacted wisdom teeth under general isoflurane gas anesthesia. There was a very high interindividual variation in tissue content, with small variation in levels found in teeth from the same patient. Pulps obtained from lower teeth without local anesthesia contained an average of 131 +/- 62 fmol/mg protein of iCGRP and 15 +/- 9 fmol/mg iSP (n = 10). With additional mandibular block anesthesia the values were 194 +/- 71 fmol/mg iCGRP (statistically significant, p = 0.0356, Mann-Whitney-rank-sum-test) and 12 +/- 3.6 fmol/mg iSP. The results suggest that local anesthesia attenuates neuropeptide release in the human dental pulp during surgical extraction.
Anesthesia, Dental↗
Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp.
The phylogeny of obligate intracellular coccoid parasites of acanthamoebae isolated from the nasal mucosa of humans was analyzed by the rRNA approach. The primary structures of the 16S and 23S rRNA molecules of one strain were determined in almost full length. In situ hybridization with a horseradish peroxidase-labeled oligonucleotide probe targeted to a unique signature site undoubtedly correlated the retrieved 16S rRNA sequence to the respective intracellular parasite. This probe also hybridized with the second strain, suggesting a close relationship between the two intracellular parasites. Comparative sequence analysis demonstrated a distinct relationship to the genus Chlamydia. With 16S rRNA similarities of 86 to 87% to the hitherto-sequenced Chlamydia species, the intracellular parasites are likely not new species of this genus but representatives of another genus in the family of the Chlamydiaceae. Consequently, it is proposed to provisionally classify the endoparasite of Acanthamoeba sp. strain Bn9 as "Candidatus Parachlamydia acanthamoebae." From an epidemiological perspective, the results suggest that small amoebae could be environmental reservoirs and vectors for a variety of potentially pathogenic bacteria including members of the Chlamydiaceae.
Acanthamoeba↗
Flow sorting of microorganisms for molecular analysis.
Not only classical cultivation-based methods but also the new molecular approaches may result in incomplete and selective information on the natural diversity of microbial communities. Flow sorting of microorganisms from environmental samples allows the deliberate selection of cell populations of interest from highly diverse systems for molecular analysis. Several cellular parameters that can be measured by flow cytometry are useful as sort criteria. Here, we report sorting of bacteria from activated sludge, lake water, and lake sediment according to differences in light scattering, DNA content, and/or affiliation to certain phylogenetic groups as assessed by fluorescein-labeled, rRNA-targeted oligonucleotide probes. Microscopy of the sorted cells showed that populations of originally low abundance could be strongly enriched by flow sorting (up to 280-fold), depending on the original abundance of the cells of interest and the type of sample sorted. The purity of the cells of interest could be further increased by repeated sorting, but this increase was limited by cell aggregation in the case of activated-sludge samples. It was possible to amplify almost full-length 16S ribosomal DNA (rDNA) fragments from sorted microbial cells by PCR, even after fixation with paraformaldehyde and in situ hybridization. Dot blot hybridization and sequencing demonstrated that most of the amplified rDNA originated from those cells that had been selected for by flow sorting. Comparative analysis of 16S rDNA sequences revealed previously unknown species of magnetotactic or activated-sludge bacteria.
Bacteria↗
Contrasting bacterial strategies to coexist with a flagellate predator in an experimental microbial assemblage.
We studied predator-induced changes within a slowly growing mixed microbial assemblage that was sustained by algal exudates in a continuous cultivation system. In situ hybridization with fluorescent monolabeled oligonucleotide probes was used for a tentative community analysis. This method also allowed us to quantify the proportions of predators with ingested bacteria of different taxonomic groups. In addition, we determined grazing rates on bacteria with fluorescently labelled prey. Bacteria belonging to the alpha and beta subdivisions of the phylum Proteobacteria ((alpha)- and (beta)-Proteobacteria, respectively) showed very different responses to the addition of a bacterivorous flagellate, Bodo saltans. Within one day, filamentous protist-inedible bacteria developed; these belonged to the (beta)-Proteobacteria and constituted between 8.7 and 34% of bacteria from this subgroup. Total abundance of (beta)-Proteobacteria decreased from 3.05 x 10(sup6) to 0.23 x 10(sup6) cells ml(sup-1), and estimated cell division rates were low. Other morphologically inconspicuous protist-edible bacteria belonging to the (alpha)-Proteobacteria were found to respond to predation by an increase in growth rate. Although these bacteria were heavily grazed upon, as on average >85% of flagellate cells had ingested (alpha)-Proteobacteria, they numerically dominated after the addition of B. saltans (mean, 1.35 x 10(sup6) cells ml(sup-1)). It was thus mainly those fast-dividing strains of (alpha)-Proteobacteria that supported the growth of the flagellate population. We conclude that bacteria in mixed assemblages can adopt at least two distinct strategies as a reaction to intense flagellate predation: to outgrow predation pressure or to develop inedible, inactive filaments. Since these strategies occurred within 24 h after the addition of the flagellate, we hypothesize that chemical stimuli released by the predator may have triggered bacterial responses.
Journal Article↗