Fecal pellet does not contain E. coli

[seqretariat]

I isolated DNA from a mouse fecal pellet from the large intestine using the QIAamp DNA Stool Mini Kit. We readily detected enterobacteriaceae using specific primers and qPCR but upon sequencing there was very little detection of enterobacteriaceae. I also tried to qPCR for E.coli but there essentially no amplification. We should really expect to see plenty of enterobacteriaceae/E. coli because the treatment we are using (DSS) has been shown to increase these populations.

Is it possible that the fecal pellet did not contain any enterobacteriaceae/E. coli especially after DSS treatment?

Could the enterobacteriaceae/E. coli populations have been nearly all localized to the mucosal surface of the large intestine?

Is it common to not detect any enterobacteriaceae/E. coli in a mouse fecal pellet?

Thanks

[asorbie]

Was this just from one mouse? Have you looked into the possibility of other intestinal enterobacteriaceae? I work with mice/16s sequencing too and although we often find E.coli it's definitely not a given that it'll show up.

[seqretariat]

This was in multiple mice. In our samples we do find the presence of Rhodospirillacea, which are in the Proteobacteria phylum, but I have seen very few papers describing them as significant components of the intestinal microbiota. I am wondering whether our downstream analysis might be affecting this outcome and identifying sequences as Rhodospirillaceae when they should be enterobacteriaceae. We are using mothur with SILVA 16S rRNA gene reference database

[asorbie]

I would guess the Rhodospirillaceae are probably environmental/kit contaminants as this family is in the alphaproteobacteria class which to my knowledge is not commonly found in intestinal environments. Which region do your 16s primers target? It's possible, although unlikely, that the primers you are using don't pick up Proteobacteria/Gammaproteobacteria very well.

[seqretariat]

We used primers 515F-806R to target the V4 region.