Whole-Cell Hybridization of Frankia Strains with Fluorescence- or Digoxigenin-Labeled, 16S rRNA-Targeted Oligonucleotide Probes

A whole-cell hybridization assay with fluorescent oligonucleotide probes derived from the 16S rRNA sequence of Brucella abortus in combination with flow cytometry has been developed. With the three fluorescent probes selected, a positive signal was observed with all the representative strains of the species and biovars of Brucella and with a total of nine differentBrucella clinical isolates. Using the B9 probe in the hybridization assay, it was possible to discriminate betweenBrucella suis biovars 2, 3, 4, and 5 and almost all the other Brucella spp. On the basis of differences in fluorescence intensities, no discrimination was established betweenBrucella spp. and other phylogenetically related microorganisms. No positive fluorescence signals were detected with any of the bacteria showing serological cross-reactions withBrucella spp. and with a total of 17 clinical isolates not belonging to the genus Brucella. These results suggest that the 16S rRNA whole-cell hybridization technique could be a valuable diagnostic tool for the detection and identification ofBrucella spp.

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Oligonucleotide Probes for the Identification of Three Algal Groups by Dot Blot and Fluorescent Whole-Cell Hybridization

Journal of Eukaryotic Microbiology ◽

10.1111/j.1550-7408.2000.tb00014.x ◽

2000 ◽

Vol 47 (1) ◽

pp. 76-84 ◽

Cited By ~ 113

Author(s):

NATHALIE SIMON ◽

LISA CAMPBELL ◽

ERLA ORNOLFSDOTTIR ◽

RENE GROBEN ◽

LAURE GUILLOU ◽

...

Keyword(s):

Oligonucleotide Probes ◽

Dot Blot ◽

Cell Hybridization ◽

Whole Cell ◽

Algal Groups

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Degradative capacities and 16S rRNA-targeted whole-cell hybridization of sulfate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil.

Applied and Environmental Microbiology ◽

10.1128/aem.62.10.3605-3613.1996 ◽

1996 ◽

Vol 62 (10) ◽

pp. 3605-3613 ◽

Cited By ~ 132

Author(s):

R Rabus ◽

M Fukui ◽

H Wilkes ◽

F Widdle

Keyword(s):

16S Rrna ◽

Crude Oil ◽

Enrichment Culture ◽

Sulfate Reducing Bacteria ◽

Cell Hybridization ◽

Whole Cell ◽

Sulfate Reducing ◽

Reducing Bacteria

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Identification of “Candidatus Thioturbo danicus,” a Microaerophilic Bacterium That Builds Conspicuous Veils on Sulfidic Sediments

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8929-8933.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8929-8933 ◽

Cited By ~ 20

Author(s):

Gerard Muyzer ◽

Esengül Yildirim ◽

Udo van Dongen ◽

Michael Kühl ◽

Roland Thar

Keyword(s):

Molecular Analysis ◽

New Genus ◽

Oligonucleotide Probes ◽

Cell Hybridization ◽

Whole Cell ◽

Appl Environ

ABSTRACT Molecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the ε-subdivision of the Proteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name “Candidatus Thioturbo danicus.”

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Rapid Identification of Bacteria in Blood Cultures by Using Fluorescently Labeled Oligonucleotide Probes

Journal of Clinical Microbiology ◽

10.1128/jcm.38.2.814-817.2000 ◽

2000 ◽

Vol 38 (2) ◽

pp. 814-817 ◽

Cited By ~ 73

Author(s):

G. J. Jansen ◽

M. Mooibroek ◽

J. Idema ◽

H. J. M. Harmsen ◽

G. W. Welling ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Bacterial Species ◽

Fluorescein Isothiocyanate ◽

Negative Control ◽

Oligonucleotide Probes ◽

Coagulase Negative Staphylococci ◽

Cell Hybridization ◽

Variable Regions ◽

Gram Negative ◽

Whole Cell

The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial species and/or genera were used: Streptococcus spp., Enterococcus faecalis, Staphylococcus aureus, coagulase-negative staphylococci (CoNS), Escherichia coli, Pseudomonas aeruginosa, and the Enterobacteriaceae family. A probe specific for the rRNAs of almost all bacteria and its complementary, reversed counterpart was used as positive and negative control, respectively. The probes were used in conjunction with a fast and simple-to-use protocol for whole-cell hybridization. This protocol yields an identification after 25 to 45 min, depending on whether the bacterium is gram positive or gram negative. A total of 182 blood samples which tested positive in a blood culture machine were investigated. All probes except for the ones for S. aureusand the CoNS showed sensitivities and specificities of 1.000. It was concluded that whole-cell hybridization is well suited for the fast screening of septic blood containing streptococci and/or enterococci or gram-negative rods.

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Whole-Cell versus Total RNA Extraction for Analysis of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide Probes in Salt Marsh Sediments

Applied and Environmental Microbiology ◽

10.1128/aem.66.7.3037-3043.2000 ◽

2000 ◽

Vol 66 (7) ◽

pp. 3037-3043 ◽

Cited By ~ 33

Author(s):

Marc E. Frischer ◽

Jean M. Danforth ◽

Michele A. Newton Healy ◽

F. Michael Saunders

Keyword(s):

Salt Marsh ◽

16S Rrna ◽

Microbial Communities ◽

Rna Extraction ◽

Oligonucleotide Probes ◽

Whole Cell ◽

Sulfate Reducing ◽

Marsh Sediments ◽

Salt Marsh Sediments ◽

Reducing Bacteria

ABSTRACT rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% Ã¯Â¿Â½ 3.7% [mean Ã¯Â¿Â½ standard deviation]) relative to direct DAPI (4′,6′-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r 2 = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.

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Discrimination of Alexandrium andersoni and A. minutum (Dinophyceae) using LSU rRNA-targeted oligonucleotide probes and fluorescent whole-cell hybridization

Phycologia ◽

10.2216/06-11.1 ◽

2007 ◽

Vol 46 (2) ◽

pp. 168-177 ◽

Cited By ~ 13

Author(s):

Nicolas Touzet ◽

Robin Raine

Keyword(s):

Oligonucleotide Probes ◽

Cell Hybridization ◽

Whole Cell ◽

Lsu Rrna

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In situ analysis of the bacterial community in the gut of the earthwormLumbricus terrestrisL. by whole-cell hybridization

Canadian Journal of Microbiology ◽

10.1139/m95-092 ◽

1995 ◽

Vol 41 (8) ◽

pp. 666-673 ◽

Cited By ~ 54

Author(s):

Kathrin Fischer ◽

Dittmar Hahn ◽

Otto Daniel ◽

Josef Zeyer ◽

Rudolf I. Amann

Keyword(s):

Bacterial Community ◽

Lumbricus Terrestris ◽

In Situ Analysis ◽

Oligonucleotide Probes ◽

Dapi Staining ◽

Cell Hybridization ◽

Whole Cell ◽

Hind Gut ◽

Number Of Bacteria

The bacterial community in the gut of the earthworm Lumbricus terrestris was analyzed by whole-cell hybridization with 16S rRNA targeted oligonucleotide probes. Whole-cell hybridization protocols using fluorescence-, peroxidase-, or digoxigenin-labeled oligonucleotide probes facilitated detection of significant fractions of bacterial cells stained with 4′,6-diamidino-2-phenylindole (DAPI) in the fore-, mid-, and hind-gut and cast of the earthworm. The application of peroxidase- and digoxigenin-labeled probes, however, was hampered by several methodological drawbacks: the requirement of enzymatic permeabilization, the diffuse images of stained cells, and the incompatibility with DAPI staining used as control. Quantitative analysis of the bacterial community was also influenced by its considerable variability in different individual earthworms. Though the number of bacteria detected by DAPI staining as well as by whole-cell hybridization with the fluorescent eubacterial probe Eub338 generally showed a significant increase in the number of bacteria towards the end of the gut, a decrease in bacterial numbers could be found in some earthworms. In situ analysis of the bacterial community in the fore-, mid-, and hind-gut of one individual earthworm by whole-cell hybridization with the fluorescent eubacterial probe Eub338 recorded 15, 30, and 25% of DAPI-stained bacteria, respectively. In the cast 37% of the bacteria were detected. Similar to counts obtained by DAPI and by whole-cell hybridization with probe Eub338, the number of bacteria belonging to the α-, β-, and γ-subgroups of proteobacteria increased significantly towards the end of the gut and remained high in the cast. While the most significant difference in the counts of bacteria belonging to the α-subgroup was obtained between the hind-gut and cast, bacterial populations of the β- and γ- subgroups of proteobacteria increased most prominently between the fore- and hind-gut.Key words: digoxigenin, fluorescent probes, in situ detection, Lumbricus terrestris, rRNA, whole-cell hybridization.

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Monitoring ofBacillus thermodenitrificansOHT-1 in compost by whole cell hybridization

Canadian Journal of Microbiology ◽

10.1139/w02-069 ◽

2002 ◽

Vol 48 (9) ◽

pp. 848-852 ◽

Cited By ~ 6

Author(s):

Masahiro Hatsu ◽

Junji Ohta ◽

Kazuhiro Takamizawa

Keyword(s):

16S Rrna ◽

Liquid Culture ◽

Oligonucleotide Probe ◽

Thermophilic Bacteria ◽

Fluorescein Isothiocyanate ◽

Rrna Sequence ◽

Cell Hybridization ◽

16S Rrna Sequence ◽

Whole Cell ◽

Growth Experiments

Thermophilic aerobic composting is a widely practiced method for the disposal of exhaust materials. We isolated a thermophilic bacteria strain from a compost sample under aerobic conditions at 60°C. On the basis of its 16S rRNA sequence and physiological characteristics, this strain was identified as Bacillus thermodenitrificans OHT-1. An 18-subunit oligonucleotide probe for 16S rRNA, labeled with fluorescein isothiocyanate, was developed for the detection of B. thermodenitrificans. Spores and vegetative cells of B. thermodenitrificans OHT-1 were detected in liquid culture and laboratory compost by whole cell hybridization using this oligonucleotide probe. The results obtained by whole cell hybridization were evaluated in growth experiments of B. thermodenitrificans OHT-1 in laboratory compost and were used to enumerate spores and vegetative cells.Key words: compost, Bacillus thermodenitrificans, 16S rRNA, whole cell hybridization.

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