Compositional variation of the human fecal microbiome in relation to azo-reducing activity: a pilot study

Background Through an arsenal of microbial enzymes, the gut microbiota considerably contributes to human metabolic processes, affecting nutrients, drugs, and environmental poisons. Azoreductases are a predominant group of microbiota-derived enzymes involved in xenobiotic metabolism and drug activation, but little is known about how compositional changes in the gut microbiota correlate with its azo-reducing activity. Results To this end, we used high-throughput 16S rRNA amplicon sequencing, with Illumina MiSeq, to determine the microbial community composition of stool samples from 16 adults with different azo-reducing activity. High azo-reducing activity positively correlated with the relative abundance of phylum Firmicutes (especially genera Streptococcus and Coprococcus) but negatively with phylum Bacteroidetes (especially genus Bacteroides). Typical variations in the Firmicutes-to-Bacteroidetes and Prevotella-to-Bacteroides ratios were observed among samples. Multivariate analysis of the relative abundance of key microbial taxa and other diversity parameters confirmed the Firmicutes proportion as a major variable differentiating high and non-azo-reducers, while Bacteroidetes relative abundance was correlated with azo-reduction, sex, and BMI. Conclusions This pilot study showed that stool samples with higher azo-reducing activity were enriched in Firmicutes but with relatively fewer Bacteroidetes. More samples and studies from different geographical areas are needed to bolster this conclusion. Better characterization of different azoreductase-producing gut microbes will increase our knowledge about the fate and differential human responses to azodye-containing drugs or orally consumed chemicals, thus contributing to efforts towards implementing microbiome testing in precision medicine and toxicology.

Reduction of azo dyes by flavin reductase from Citrobacter freundii A1

<em>Citrobacter freundii </em>A1 isolated from a sewage treatment facility was demonstrated to be able to effectively decolorize azo dyes as pure and mixed culture. This study reports on the investigation on the enzymatic systems involved. An assay performed suggested the possible involvement of flavin reductase (Fre) as an azo reductase. A heterologouslyexpressed recombinant Fre from <em>C. freundii </em>A1 was used to investigate its involvement in the azo reduction process. Three model dyes were used, namely Acid Red 27 (AR27), Direct Blue 15 (DB15) and Reactive Black 5 (RB5). AR27 was found to be reduced the fastest by Fre, followed by RB5, and lastly DB15. Redox mediators nicotinamide adenine dinucleotide (NADH) and riboflavin enhance the reduction, suggesting the redox activity of the enzyme. The rate and extent of reduction of the model dyes correlate well with the reduction potentials (<em>E</em>_p). The data presented here strongly suggest that Fre is one of the enzymes responsible for azo reduction in <em>C. freundii </em>A1, acting via an oxidation-reduction reaction.