scholarly journals The Mucin Degrader Akkermansia muciniphila Is an Abundant Resident of the Human Intestinal Tract

2007 ◽  
Vol 74 (5) ◽  
pp. 1646-1648 ◽  
Author(s):  
Muriel Derrien ◽  
M. Carmen Collado ◽  
Kaouther Ben-Amor ◽  
Seppo Salminen ◽  
Willem M. de Vos
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Intestinal Tract ◽  
Akkermansia Muciniphila ◽  
Human Intestinal Tract

ABSTRACT A 16S rRNA-targeted probe, MUC-1437, was designed and validated in order to determine the presence and numbers of cells of Akkermansia muciniphila, a mucin degrader, in the human intestinal tract. As determined by fluorescent in situ hybridization, A. muciniphila accounted more than 1% of the total fecal cells and was shown to be a common bacterial component of the human intestinal tract.

scholarly journals Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ

1999 ◽  
Vol 65 (4) ◽  
pp. 1746-1752 ◽  
Author(s):  
Cleber C. Ouverney ◽  
Jed A. Fuhrman
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Microbial Communities ◽  
Fluorescent In Situ Hybridization ◽  
Amino Acid Uptake ◽  
Cell Types ◽  
Black Box ◽  
Acid Uptake ◽  
Substrate Uptake

ABSTRACT We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class Proteobacteria(α-Proteobacteria) and of the Cytophaga-Flavobacteriumgroup obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (∼60% of the universal probe-labeled cells and ∼50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.

Novel Nitrospira-like bacteria as dominant nitrite-oxidizers in biofilms from wastewater treatment plants: diversity and in situ physiology

2000 ◽  
Vol 41 (4-5) ◽  
pp. 85-90 ◽  
Author(s):  
H. Daims ◽  
P.H. Nielsen ◽  
J.L. Nielsen ◽  
S. Juretschko ◽  
M. Wagner
Keyword(s):  
Wastewater Treatment ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Wastewater Treatment Plants ◽  
Combined Application ◽  
Fundamental Understanding ◽  
Unculturable Bacteria ◽  
Rrna Sequences

The frequency and distribution of putatively nitrite-oxidizing, Nitrospira- like bacteria in nitrifying biofilms from two reactors receiving wastewater with different ammonia and salt concentrations were observed by fluorescent in situ hybridization. For this purpose, new 16S rRNA-directed oligonucleotide probes targeting the bacterial phylum Nitrospira and the three main lineages within this phylum were developed and evaluated. The diversity of Nitrospira-like bacteria in the reactors was additionally investigated by retrieval and comparative analysis of full 16S rRNA sequences from the biofilms. We found that, despite of the differences in the influent composition, Nitrospira-like bacteria form dominant populations in both reactors. In addition, first insights into the physiology of these still unculturable bacteria were obtained by the incubation of active biofilm samples with radioactively labeled substrates followed by the combined application of fluorescent in situ hybridization and microautoradiography. The results are discussed in consideration of the frequently observed dominance of Nitrospira-like bacteria in nitrifying bioreactors. Consequently, high priority should be assigned to future studies on the ecology and physiology of these organisms in order to increase our fundamental understanding of nitrogen cycling and to enable knowledge-driven future improvements of nitrifying wastewater treatment plants.

16S rRNA fluorescent in situ hybridization of human colon and terminal ileum biopsies: A mucus-adherent bacterial flora does not exist

2001 ◽  
Vol 120 (5) ◽  
pp. A706
Author(s):  
Laurens A. Van der Waaij ◽  
Hermie J.M. Harmsen ◽  
Mohsen Madjipour ◽  
Frans G.M. Kroese ◽  
Hendrik M. Van Dullemen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Terminal Ileum ◽  
Fluorescent In Situ Hybridization ◽  
Bacterial Flora ◽  
Human Colon

scholarly journals Intestinal Integrity and Akkermansia muciniphila, a Mucin-Degrading Member of the Intestinal Microbiota Present in Infants, Adults, and the Elderly

2007 ◽  
Vol 73 (23) ◽  
pp. 7767-7770 ◽  
Author(s):  
M. Carmen Collado ◽  
Muriel Derrien ◽  
Erika Isolauri ◽  
Willem M. de Vos ◽  
Seppo Salminen
Keyword(s):  
Early Life ◽  
Intestinal Tract ◽  
The Elderly ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Akkermansia Muciniphila ◽  
Intestinal Integrity ◽  
The 16S Rrna Gene ◽  
Human Intestinal Tract

ABSTRACT Fluorescence in situ hybridization and real-time PCR analysis targeting the 16S rRNA gene of Akkermansia muciniphila were performed to determine its presence in the human intestinal tract. These techniques revealed that an A. muciniphila-like bacterium is a common member of the human intestinal tract and that its colonization starts in early life and develops within a year to a level close to that observed in adults (108 cells/g) but decreases (P < 0.05) in the elderly.

scholarly journals Feasibility of Transferring Fluorescent In Situ Hybridization Probes to an 18S rRNA Gene Phylochip and Mapping of Signal Intensities

2008 ◽  
Vol 74 (9) ◽  
pp. 2814-2821 ◽  
Author(s):  
Katja Metfies ◽  
Linda K. Medlin
Keyword(s):  
In Situ Hybridization ◽  
Secondary Structure ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Rrna Molecule ◽  
The 16S Rrna Gene

ABSTRACT DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.

16S rRNA fluorescent in situ hybridization of human colon and terminal ileum biopsies: A mucus-adherent bacterial flora does not exist

2001 ◽  
Vol 120 (5) ◽  
pp. A706-A706
Author(s):  
L VANDERWAAIJ ◽  
H HARMSEN ◽  
M MADJIPOUR ◽  
F KROESE ◽  
H VANDULLEMEN ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Terminal Ileum ◽  
Fluorescent In Situ Hybridization ◽  
Bacterial Flora ◽  
Human Colon

scholarly journals Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization

2002 ◽  
Vol 68 (1) ◽  
pp. 356-364 ◽  
Author(s):  
Tsukasa Ito ◽  
Jeppe L. Nielsen ◽  
Satoshi Okabe ◽  
Yoshimasa Watanabe ◽  
Per H. Nielsen
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Electron Acceptor ◽  
Fluorescent In Situ Hybridization ◽  
Sulfate Reducing Bacteria ◽  
Substrate Uptake ◽  
Sulfate Reducing ◽  
Phylogenetic Identification ◽  
Reducing Bacteria

ABSTRACT We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [14C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H2-utilizing and 14CO2-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H2-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H2 utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.

Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

1998 ◽  
Vol 64 (9) ◽  
pp. 3336-3345 ◽  
Author(s):  
Alison H. Franks ◽  
Hermie J. M. Harmsen ◽  
Gerwin C. Raangs ◽  
Gijsbert J. Jansen ◽  
Frits Schut ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Blot Hybridization ◽  
Dry Weight ◽  
Oligonucleotide Probes ◽  
Fecal Flora ◽  
Dot Blot ◽  
Clostridium Histolyticum

ABSTRACT Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the speciesBacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and theClostridium lituseburense groups. Another probe was designed for the genera Streptococcus andLactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectalegroup. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 1010 cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectalegroup-specific probe detected a mean of 7.2 × 1010cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, andStreptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 107 to 7 × 108 per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.

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