Inter-correlated gut microbiota and SCFAs changes upon antibiotics exposure links with rapid body-mass gain in weaned piglet model

 The risk of overweight or obesity in association with early exposure of antibiotics remains an important public issue for health-care of children

• Low-dose antibiotics (LDA) have been widely used to enhance growth rate of pigs, providing a good animal model to study the underlying mechanism.
• In present study, 28 female piglets, weaned at 21 d, were randomly classified into two groups, receiving either a control diet or a diet supplemented with LDA for 4 weeks.

Gut microbiota is involved in the digestion of proteins, lipids and carbohydrates in small intestine, and fermenting indigestible polysaccharides into low-molecular-weight metabolites, e.g. short-chain fatty acids (SCFAs).

• SCFAs represent 10% of the human daily energy intake and modulate crucial biological process, such as hepatic gluconeogenesis and cholesterol biosynthesis.
• The sub-therapeutic use of antibiotics in animal feed promotes feed intake and rapid growth.

Animals and diets

• Twenty-eight female piglets with body weight at 6.50±0.20 kg were weaned at 21 day-old-age, and randomly divided into two groups, receiving a basic diet and diet supplemented with LDA (n=14)
• Feed supplied to the piglets was recorded daily
• Individual piglet BW and feed disappearance was measured weekly to calculate average daily gain (ADG) and average daily feed intake (ADFI).
• For testing the digestibility of nutrients, during the third week, chromium oxide (Cr2O3) was added in each diet at 3 g/kg

Targeted metabolomics of SCFAs detection

• The optimized UPLC-QTRAP-MS/MS method was employed in our study with some modifications.
• With a single set of optimized reaction conditions, 3-nitrophenylhydrazine (3NPH) was employed for pre-analytical derivatization to convert SCFs as well as their 3NPH derivatives, which showed excellent chemical stability.

Quantitative PCR (qPCR)

• Total DNA was extracted from gut content for each sample using the CTAB/SDS method.
• Each PCR reaction mixture (20 μl) contained 5 μl of template DNA, 10 μl SuperReal PreMix Plus (SYBR Green, TIANGEN, FP205), and 1.5 μl each primer (10 μM).
• The qPCR reactions were performed in triplicate under thermal cycler conditions, with 39 cycles of 10 s at 95°C, 30 s at 55°C and 32 s at 72°C in a CFX Connect™ Real-Time PCR Detection System.

DNA extraction, PCR amplification of 16S rRNA gene, amplicon sequence and sequence data processing

• Total genome DNA was extracted from contents of ileum and colon using the CTAB/SDS method.
• DNA concentration measured by Qubit 3.0 Fluorometer and purity were monitored on 1% agarose gels.

16S rRNA amplicon sequencing data analysis

• Sequences were quality-filtered and de-noised using the Divisive Amplicon Denoising Algorithm 2 (DADA2)
• Taxonomy was assigned using the 99% identity SILVA (release 119) V3-V4 classifier
• All the ribosomal sequence variants (RSVs) were identified as features across all samples without clustering
• Differences in beta diversity were identified using Analysis of Similarity (ANOSIM) and effect size was indicated by an R-value
• Significant correlations were indicated with an absolute Pearson's correlation coefficient above 0.50 and a P-value under 0.05
• Community structure difference based on beta diversity was visualized using principal coordinate analysis (PCoA) by R package VEGAN

RNA isolation, quantification, library preparation, sequencing and transcriptome analysis of intestinal tissues

• Total RNAs of colon tissues were extracted by QIAGEN RNeasy Protect Animal Blood Kit (Qiagen, Germany) and RNA degradation and contamination were monitored on 1% agarose gels.
• RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit RNA Assay Kit in Qubeit 2.0 Flurometer (Life Technologies, Calif., USA). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, and DNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-).
• Sequencing libraries were generated using NEBNext UltraTM Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations.

Validation of DEG expression by real-time PCR

• The total RNA was reversely transcribed by using PrimeScript RT reagent Kit with gDNA Eraser (Takara)
• Real time PCR reactions were performed as follows
• One cycle (42°C 5 min); one cycle (95°C 10 s); forty cycles (95%C 5 s, 60°C 34 s)+ one cycle (<1 cycle) of each gene was run in duplicate and three times for obtaining reliable amplification efficiency values
• All RT-PCR target gene expression was normalized to the expression of β-action and the relative quantification was analyzed using the 2ΔΔCt method

Growth performance and nutrients digestibility

• After 4 weeks of treatment, the growth-promoting effect of LDA was observed, as indicated by the increased ADG, ADFI, and ADFI (P=.026).

SCFAs and SBCFAs

• Regardless of LDA treatment, there were significantly higher (P<.001) levels of these in the content of colon than in ileum.

Bacteria load and microbial diversity

• The LDA group had significantly reduced total bacterial load in ileum content (2.59×1011 vs. 8.23×1013 16S rRNA copies per gram) compared to CON group
• Relative to CON, LDA significantly reduced bacterial loads in colon contents
• Alpha diversity analysis was performed for each group to compare the species diversity within each microbial community
• However, no clear difference of alpha diversity was observed between the LDA and CON groups

Bacterial abundance and functional implication

• Firmicutes was the dominant phylum both in colon and ileum contents.
• The abundance of Bacteroidetes was decreased (P<.001) in the colon content of LDA group in comparison with CON group (Fig. 3a).
• At the genus level, most of the annotated features belonged to Lachnospiraceae spp., Lactobacillus, Ruminococcaceae, Clostridium, and Actinobacteria in colon, and Turicibacter, Actinococcus, Escherichia-Shigella, Turicibia, and Lacticibacter
• A total of 28 significantly different genera with average relative abundance >0.01% at least in one group was identified.

Alteration of SCFAs production associates with gut microbiota shift

• LDA exposure shifted the genera to highly correlate with the three SCFA, as illustrated by the co-occurrence networks (Fig. 4).
• In the control group, the positively correlated genera were mainly comprised of Prevotellaceae, Spirochaetaceae and Methanobacteriaceae.
• The absolute Pearson’s coefficients for each of these genera had significant correlations (absolute Pearson's coefficients >0.50 and P<.05) with the abundance of each genus.

Gut microbiota shift and SCFAs alteration associate with colonic gene expressions

• We obtained 45.41±7.89 million clean reads per sample, of which 76.65±5.19% could be aligned to the pig reference genome (Supplementary file 1: Table S12).
• Fifty-two DEGs were identified (FC>2, P<.05), in which 49 were up-regulated upon LDA exposure.
• GO enrichment analysis indicated 16 DEGs are involved in biological process of "cell proliferation or normal development", "immune response", "nervous system".
• In addition, we found genes in relation with neuron cells, including FGF14, NRG1, NTRK2 and EFNA5.

Discussion The early exposure to antibiotics has been shown to increase risk of overweight or obesity later in childhood

• The growth-promoting effect of LDA in pig model mimics the similar consequences in human-being
• LDA markedly decreased bacterial loads in ileum but not colon, probably due to that the total bacterial load in the colon content is much higher than in the ilesum content and LDA was not sufficient to change the absolute numbers of bacterial cells
• These results highlighted the significance of colon as a major organ for the nutritional effects of microbiota, as indicated by the markedly increased F/B index and the members of the Firmicutes and Actinobacteria phyla include species degrading the dietary carbohydrates
• Increased abundance of Methanosphaera species in colon upon LDA exposure has been associated with obesity [36] and host energy extraction from indigestible polysaccharides [37]

Reference

10.1016/j.jnutbio.2019.108246