The response of the mouse microbiome to ketogenic diet in a colorectal cancer model with a humanized microbiome (GF mice) analyzed by 16S gene sequencing. null

In this study, the beneficial effect of ketogenic diet in the context of colorectal cancer was analyzed using a CRC mouse model with a humanized gut microbiome. For this purpose, GF mice received a mixed microbiome generated from five human fecal samples from healthy donors by cecal gavage and were either fed a ketogenic-diet or standard diet. In the ketogenic diet group reduced colonic tumor burden and distinct changes in fecal microbial and metabolic profiles were observed, which were partly microbiome mediated as demonstrated by a cecal microbiome transfer experiment. To analyze changes in microbiome composition over time, fecal samples were collected throughout the experiment and were used for 16S amplicon sequencing. Briefly, DNA was extracted using the DNeasy PowerSoil DNA Isolation Kit (Qiagen), according to the kit manual, except that samples were homogenized using the TissueLyzerII for five minutes at 40Hz instead of intense vortexing followed by determination of concentrations and purities of the extracted DNA samples using a Nanodrop 2000 spectrophotometer purification and concentration using the DNA Clean and Concentrator Kit (Zymo Research). Prepared libraries were quantified using the Qubit® dsDNA BR Assay Kit (Thermofisher Scientific) and Qubit® 3 Fluorometer (ThermoFisher Scientific) and sample quality was assessed using the Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent Technologies). Finally, 5 ng of DNA extracted was subjected to amplicon sequencing of the V4 region of 16S rRNA genes using primers 515F and 805R on a MiSeq instrument (Illumina) using 2×300 base pair read lengths.