PMID- 27665238
OWN - NLM
STAT- MEDLINE
DCOM- 20170407
LR  - 20170407
IS  - 1618-0984 (Electronic)
IS  - 0723-2020 (Linking)
VI  - 39
IP  - 7
DP  - 2016 Oct
TI  - Flow cytometric sorting of fecal bacteria after in situ hybridization with 
      polynucleotide probes.
PG  - 464-475
LID - S0723-2020(16)30088-1 [pii]
LID - 10.1016/j.syapm.2016.08.005 [doi]
AB  - The gut microbiome represents a key contributor to human physiology, metabolism, 
      immune function, and nutrition. Elucidating the composition and genetics of the 
      gut microbiota under various conditions is essential to understand how microbes 
      function individually and as a community. Metagenomic analyses are increasingly 
      used to study intestinal microbiota. However, for certain scientific questions it 
      is sufficient to examine taxon-specific submetagenomes, covering selected 
      bacterial genera in a targeted manner. Here we established a new variant of 
      fluorescence in situ hybridization (FISH) combined with fluorescence-activated 
      cell sorting (FACS), providing access to the genomes of specific taxa belonging 
      to the complex community of the intestinal microbiota. In contrast to standard 
      oligonucleotide probes, the RNA polynucleotide probe used here, which targets 
      domain III of the 23S rRNA gene, extends the resolution power in environmental 
      samples by increasing signal intensity. Furthermore, cells hybridized with the 
      polynucleotide probe are not subjected to harsh pretreatments, and their genetic 
      information remains intact. The protocol described here was tested on 
      genus-specifically labeled cells in various samples, including complex fecal 
      samples from different laboratory mouse types that harbor diverse intestinal 
      microbiota. Specifically, as an example for the protocol described here, RNA 
      polynucleotide probes could be used to label Enterococcus cells for subsequent 
      sorting by flow cytometry. To detect and quantify enterococci in fecal samples 
      prior to enrichment, taxon-specific PCR and qPCR detection systems have been 
      developed. The accessibility of the genomes from taxon-specifically sorted cells 
      for subsequent molecular analyses was demonstrated by amplification of functional 
      genes.
CI  - Copyright (c) 2016 Elsevier GmbH. All rights reserved.
FAU - Bruder, Lena M
AU  - Bruder LM
AD  - Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, 
      Freising-Weihenstephan, Germany. Electronic address: lena.bruder@tum.de.
FAU - Dorkes, Marcel
AU  - Dorkes M
AD  - Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, 
      Freising-Weihenstephan, Germany.
FAU - Fuchs, Bernhard M
AU  - Fuchs BM
AD  - Max-Planck-Institut fur Marine Mikrobiologie, Abteilung Molekulare Okologie, 
      Bremen, Germany.
FAU - Ludwig, Wolfgang
AU  - Ludwig W
AD  - Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, 
      Freising-Weihenstephan, Germany.
FAU - Liebl, Wolfgang
AU  - Liebl W
AD  - Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, 
      Freising-Weihenstephan, Germany. Electronic address: wliebl@wzw.tum.de.
LA  - eng
PT  - Journal Article
DEP - 20160913
PL  - Germany
TA  - Syst Appl Microbiol
JT  - Systematic and applied microbiology
JID - 8306133
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Bacterial)
RN  - 0 (RNA, Ribosomal, 16S)
RN  - 0 (RNA, Ribosomal, 23S)
SB  - IM
MH  - Animals
MH  - Enterococcus faecalis/*genetics
MH  - Feces/*microbiology
MH  - Flow Cytometry/*methods
MH  - Gastrointestinal Microbiome/*genetics
MH  - Germ-Free Life
MH  - In Situ Hybridization, Fluorescence
MH  - Mice
MH  - Nucleic Acid Hybridization/*methods
MH  - Oligonucleotide Probes/*genetics
MH  - RNA, Bacterial/genetics
MH  - RNA, Ribosomal, 16S/genetics
MH  - RNA, Ribosomal, 23S/genetics
OTO - NOTNLM
OT  - Enterococcus
OT  - FACS
OT  - FISH
OT  - Gut microbiome
OT  - Polynucleotide probe
OT  - Specific cell enrichment
EDAT- 2016/09/26 06:00
MHDA- 2017/04/08 06:00
CRDT- 2016/09/26 06:00
PHST- 2016/05/06 00:00 [received]
PHST- 2016/08/18 00:00 [revised]
PHST- 2016/08/19 00:00 [accepted]
PHST- 2016/09/26 06:00 [pubmed]
PHST- 2017/04/08 06:00 [medline]
PHST- 2016/09/26 06:00 [entrez]
AID - S0723-2020(16)30088-1 [pii]
AID - 10.1016/j.syapm.2016.08.005 [doi]
PST - ppublish
SO  - Syst Appl Microbiol. 2016 Oct;39(7):464-475. doi: 10.1016/j.syapm.2016.08.005. 
      Epub 2016 Sep 13.