Preliminary experiments for ARDRA validation on flora associated with intestinal mucosa

Abstract

The human intestinal tract harbours a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human faeces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Almost all studies used faecal samples, and there is little data on bacteriology of mucosa-associated bacteria of the intestinal tract. We examined the procedure to extract DNA from intestinal tissue in sufficient quantity and quality for PCR amplification. Intestinal mucosa from germ-free and conventional rats was studied. Intestinal samples were washed to remove nonadherent bacteria, and the mucosa scraped off with a sterile scalpel. Microorganisms and cells were lysed by three enzymatic treatments for total community genomic DNA isolation. Released DNA was purified by spin-column and tissue had to be stored for 24 h in a reduced transport media for PCR amplification. Universal and bacterial oligonucleotides complementary to conserved regions in the 16S rDNA of 180 organisms were evaluated, before being used for PCR amplification. All these primers contain mismatches with some organisms which may influence PCR amplification. The 16S rDNA PCR with universal primers hybridises with a mitochondrial gene of rat, producing a 550 bp fragment which contaminates 16S rDNA fragments. Fifteen cycles of amplifications were performed to preserve the biodiversity of the sample. Theoretical 16S rDNA fragments with universal primers were calculated by a restriction fragment length analysis termed ARDRA (amplified rDNA restriction analysis). Three enzymes were used: HaeIII, RsaI and TaqI. Clustering was performed after separate restriction analysis with these enzymes. An example of this method was carried out after PCR amplification of the 16S rDNA fragment of five strains isolated from a jejunal sample by microbiology techniques. Further investigations on a larger number of strains will permit us to validate more precisely this method.