Extensive set of 16S rRNA-based probes for detection of bacteria in human feces

Abstract

For the detection of six groups of anaerobic bacteria in human feces, we designed seven new 16S rRNA-based oligonucleotide probes. This set of probes extends the current set of probes and gives more data on the composition of the human gut flora. Probes were designed for Phascolarctobacterium and relatives (Phasco741), Veillonella (Veil223), Eubacterium hallii and relatives (Ehal1469), Lachnospira and relatives (Lach571), and Eubacterium cylindroides and relatives (Ecyl387), and two probes were designed for Ruminococcus and relatives (Rbro730 and Rfla729). The hybridization conditions for the new probes were optimized for fluorescent in situ hybridization, and the probes were validated against a set of reference organisms. The probes were applied to fecal samples of 11 volunteers to enumerate their target bacterial groups. The Phasco741 and Veil223 probes both detected average numbers below 1% of the total number of bacteria as determined with the bacterial kingdom-specific Bact338 probe. The Ecyl387 probe detected about 1.4%, the Lach571 and Ehal1469 probes detected 3.8 and 3.6%, respectively, and a combination of the Rbro730 and Rfla729 probes detected 10.3%. A set of 15 probes consisting of probes previously described and those presented here were evaluated in hybridization with the fecal samples of the same volunteers. Together, the group-specific probes detected 90% of the total bacterial cells.

FIG. 1.

Phylogenetic tree based on 16S rRNA sequences illustrating the target groups of the newly described probes and the other probes of the extensive gut microbiota probe set. The tree shows the relationship between the major genera, groups, and organisms known to be present in the human gut. The species or clone sequences with their corresponding accession numbers are the target organisms that have a full match with the newly described oligonucleotide probes depicted behind the vertical bars on the right. Boxes and group names in boldface indicate that the target groups of the probes have been described previously. The sizes of the boxes reflect the number of sequences used in the tree construction, and their shape reflects the phylogenetic depth of the corresponding groups. The size bar at the bottom represents 10% sequence divergence.

FIG. 2.

FISH experiments with the newly designed probes on fecal samples from different volunteers. (A) Epifluorescent images of a hybridization with the Ecyl387 probe specific for the E. cylindroides group showing small fluorescent rods. (B) Hybridization with a combination of the Rbro729 and the Rfla730 probes specific for bacteria of the Ruminococcus group; the image shows one rod and different cocci. (C) Hybridization with the Veil223 probe specific for Veillonella showing small cocci. (D) Hybridization with the Phasco741 probe detecting rods of the Phascolarctobacterium group. (E and F) Two images of different fecal samples hybridized with the Lach571 probe specific for the Lachnospira group showing big oval dividing rods and one thin rod (E) or only thin rods (F). (G) Hybridization with the Ehal1469 probe showing rods of the E. hallii group. Bar, 5 μm.