Separation of bacteria of the Clostridium leptum subgroup from the human colonic microbiota by fluorescence-activated cell sorting or group-specific PCR using 16S rRNA gene oligonucleotides.
Molecular methods based on 16S rRNA gene sequence analyses have shown that bacteria of the Clostridium leptum subgroup are predominant in the colonic microbiota of healthy humans; this subgroup includes bacteria that produce butyrate, a source of energy for intestinal epithelial cells. To improve our understanding of the species within this important group, separation methods using fluorescence-activated cell sorting (FACS) and specific PCR were combined with 16S rRNA gene sequence analyses. FACS was developed for bacteria labelled in situ with two rRNA oligonucleotide probes, namely EUB338-FITC for total bacteria and Clep866-CY5/cp or Fprau645-CY5 for bacteria of the C. leptum subgroup. Bacterial cell sorting allowed a selective recovery of members of the C. leptum subgroup from the human microbiota with efficiencies as high as 95%. Group-specific PCR amplification of the C. leptum subgroup was developed, and temporal thermal gradient gel electrophoresis showed host-specific profiles with low complexity, with a sharing of common bands between individuals and bands stable over 2 months for the same individual. A library of 16S rRNA gene cloned sequences (106 sequences) was prepared with DNA obtained from both separation methods, and 15 distinct phylotypes were identified, among which 10 have no cultivable or currently cultivated representative in reference collections.
Lay
Christophe
C
INRA, UR910, Unité d'Ecologie et Physiologie du Système Digestif, Jouy en Josas, France.
Doré
Joël
J
Rigottier-Gois
Lionel
L
D003013:Clostridium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D003106:Colon / Q000382:microbiology*; D004269:DNA, Bacterial / Q000235:genetics; D005243:Feces / Q000382:microbiology; D005434:Flow Cytometry; D020459:Genes, rRNA; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D009841:Oligonucleotides; D012336:RNA, Ribosomal, 16S