Lincomycin-induced Microbial Alteration And Their Relationship With Host Metabolic Profiles In Rats

ObjectiveTo probe the relationship between microbiome and the host metabolic changes, we analyzed the metabonomic and microbiological response of rats exposed to lincomycin by an integrated approach combining 16S rRNA gene sequencing and 1H NMR-based metabolomics profiling. This study developed a systematic and comprehensive analysis to correlate the gut microbiota variation and metabolites, which provides new insights for understanding the contributions of gut microbiota in human health and disease.Methods1. Lincomycin-induced microbial profiles changes using PCR-DGGE analysisWe used lincomycin to induce a gut microbiota imbalanced model. In addition, polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) of the V3 region of the 16S ribosomal ribonucleic acid (16S rRNA) sequences were employed for description of the diversity of the gut microbiome after lincomycin treatment. In the same time, PCR was performed using specific primers (Clostridium leptum subgroup or the Bacteroides spp.) based on the extracted DNA and the PCR products that were electrophoresed in DGGE gels.2. Lincomycin-induced urinary and faecal metabolites changes by NMR analysisThe proton spectra of faeces and urine were collected on a Broker AⅧ 600 MHz spectrometer. Before pattern recognition analysis, all spectra were phased and baseline-corrected, referenced to the TSP and normalized to the total sum of all integral regions. To screen metabolites with striking changes contributing to the separations between control and lincomycin groups, multiple components analysis such as PCA, PLS-DA, OPLS-DA were performed. Finally, the resultant metabolites were searched against Human Metabolome Database (HMDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG) followed by metabolic pathway or function analysis.3. Integration of 1H NMR spectral data and DGGE microbial dataTo reveal the potential correlations between gut microbiome and metabolites, NMR and DGGE data of faeces were correlated by using Orthogonal Projection to Latent Structure (OPLS) regression and Pearson’s correlation coefficientResults1. After 14-day lincomycin treatment, the bacterial diversity of lincomycin was found to impacted the abundance level and diversity of gut microbiota compared with the control group. Levels of Bamesiella and Prevotella decreased sharply, whereas level of Ctostridium cluster ⅩⅣa increased slightly. Twelve differential bands were identified by cloning and sequencing of 16S rRNA gene V3 regions. The sequences are deposited in the GenBank with access numbers of KP 666050— 6660612. Lincomycin exposure resulted in decreased levels of hippurate, short chain fatty acids (SCFAs) and primary bile acids and increased levels of choline and oligosaccharides. The key altered metabolites were widely distributed across the mammalian-microbial metabolic system involved in gut microbiome metabolism, energy metabolism, bile acid enterohepatic recycling and nucleic acids synthesis3. There was a strong correlation between fecal metabolites and genus of Escherichia coli, Barnesiella and Prevotella. The genus of Escherichia coli exhibited positive correlations with choline, tyrosine, TCA and uridine and reverse correlations with α-ketoisovalerate, glycine, propionate and acetate. Within the identified three Barnesiella genera, there were positive correlations with the levels of SCFAs, and negative correlations with urocanate and isoleucine.ConclusionWe developed a global analysis to study the correlation between the gut microbiota and host metabolism, showing that lincomycin not only changes the composition and abundance of bacteria but also change the host metabolism. In addition, the key functional members of gut microbiome can modulate the host specific metabolic pathways. Advances in technology in both metabolic phenotyping and microbial profiling methods have improved our ability to derive correlations between microbial and metabolic phenotypes, which are of particular important for understanding the function of gut micobiota in human health and disease.