High taxonomic level fingerprint of the human intestinal microbiota by ligase detection reaction--universal array approach

Abstract

Background: Affecting the core functional microbiome, peculiar high level taxonomic unbalances of the human intestinal microbiota have been recently associated with specific diseases, such as obesity, inflammatory bowel diseases, and intestinal inflammation.

Results: In order to specifically monitor microbiota unbalances that impact human physiology, here we develop and validate an original DNA-microarray (HTF-Microbi.Array) for the high taxonomic level fingerprint of the human intestinal microbiota. Based on the Ligase Detection Reaction-Universal Array (LDR-UA) approach, the HTF-Microbi.Array enables specific detection and approximate relative quantification of 16S rRNAs from 30 phylogenetically related groups of the human intestinal microbiota. The HTF-Microbi.Array was used in a pilot study of the faecal microbiota of eight young adults. Cluster analysis revealed the good reproducibility of the high level taxonomic microbiota fingerprint obtained for each of the subject.

Conclusion: The HTF-Microbi.Array is a fast and sensitive tool for the high taxonomic level fingerprint of the human intestinal microbiota in terms of presence/absence of the principal groups. Moreover, analysis of the relative fluorescence intensity for each probe pair of our LDR-UA platform can provide estimation of the relative abundance of the microbial target groups within each samples. Focusing the phylogenetic resolution at division, order and cluster levels, the HTF-Microbi.Array is blind with respect to the inter-individual variability at the species level.

Figure 1

SSU rRNA based phylogenetic tree of the 16S rRNA sequences of the HTF-Microbi.Array positive set. For each node we report the number of sequences used from our ARB 16S rRNA sequence database. The triangles dimension is proportional to the number of sequences clustered together. The phylogenetic tree was obtained by using the neighbour-joining algorithm for the sequence alignment in ARB software.

Figure 2

Complex mix of 16 rRNA amplicons. LDR-universal array experiments carried out on a complex mix of 16 rRNA amplicons obtained from six members of the human intestinal microbiota: B. cereus, L. casei, B. adolescentis, R. albus, Prevotella, Y. enterocolitica. Amplicons were tested in a concentration ranging from 0.7 to 75 fmol. Blue stars over the fluorescence bars indicate the probes that gave a positive response with a P < 0.01. Red dots indicate that one or two replicates out of four for each ZipCode were excluded because of having an IF < 2.5 times the average of the spots.

Figure 3

Phylogenetic fingerprints. Cluster analysis of the phylogenetic fingerprint of 16 faecal samples from 8 young adults. Response of each of the HTF-Microbi.Array probes for what concerns presence/absence of the target group is showed: positive response in red (P < 0.01), negative responses in blue (P > 0.01). Gary lines below the samples indicate adjacent replicated LDR of the same sample.

Figure 4

IF relative contribution. For each sample the entire HTF-Microbi.Array probe set was considered and their relative IF contribution was calculated as percentage of the total IF. Sub-probes were excluded and for each subject data from two separate LDR-universal array experiments were taken onto consideration. The averaged IF from both the LDR-Universal Array experiments was considered. The principal intestinal groups of major mutualistic symbionts are indicated: Bacteroides/Prevotella (B/P) blue, Clostridium cluster IV (Cl.IV) green, Clostridium cluster IX (Cl.IX) brown, Clostridium cluster XIVa (Cl.XIVa) dark brown. Lactobacillus, B. clausii, B. subtilis, Fusobacterium and Cyanobacteria are grouped as minor mutualistic symbionts (minor) indicated in yellow. Proteus, Yersinia and E. faecalis are grouped as opportunistic pathogens (opp) in red.