Microbiota of the Gut-Lymph Node Axis: Depletion of Mucosa-Associated Segmented Filamentous Bacteria and Enrichment of Methanobrevibacter by Colistin Sulfate and Linco-Spectin in Pigs

Abstract

Microorganisms are translocated from the gut to lymphatic tissues via immune cells, thereby challenging and training the mammalian immune system. Antibiotics alter the gut microbiome and consecutively might also affect the corresponding translocation processes, resulting in an imbalanced state between the intestinal microbiota and the host. Hence, understanding the variant effects of antibiotics on the microbiome of gut-associated tissues is of vital importance for maintaining metabolic homeostasis and animal health. In the present study, we analyzed the microbiome of (i) pig feces, ileum, and ileocecal lymph nodes under the influence of antibiotics (Linco-Spectin and Colistin sulfate) using 16S rRNA gene sequencing for high-resolution community profiling and (ii) ileocecal lymph nodes in more detail with two additional methodological approaches, i.e., cultivation of ileocecal lymph node samples and (iii) metatranscriptome sequencing of a single lymph node sample. Supplementation of medicated feed showed a local effect on feces and ileal mucosa-associated microbiomes. Pigs that received antibiotics harbored significantly reduced amounts of segmented filamentous bacteria (SFB) along the ileal mucosa (p = 0.048; 199.17-fold change) and increased amounts of Methanobrevibacter, a methanogenic Euryarchaeote in fecal samples (p = 0.005; 20.17-fold change) compared to the control group. Analysis of the porcine ileocecal lymph node microbiome exposed large differences between the viable and the dead fraction of microorganisms and the microbiome was altered to a lesser extent by antibiotics compared with feces and ileum. The core microbiome of lymph nodes was constituted mainly of Proteobacteria. RNA-sequencing of a single lymph node sample unveiled transcripts responsible for amino acid and carbohydrate metabolism as well as protein turnover, DNA replication and signal transduction. The study presented here is the first comparative study of microbial communities in feces, ileum, and its associated ileocecal lymph nodes. In each analyzed site, we identified specific phylotypes susceptible to antibiotic treatment that can have profound impacts on the host physiological and immunological state, or even on global biogeochemical cycles. Our results indicate that pathogenic bacteria, e.g., enteropathogenic Escherichia coli, could escape antibiotic treatment by translocating to lymph nodes. In general ileocecal lymph nodes harbor a more diverse and active community of microorganisms than previously assumed.

Keywords: 16S rRNA gene; antibiotics; gut microbiota; ileum; lymph nodes; metatranscriptome; microbiome; segmented filamentous bacteria.

FIGURE 1

Beta diversity analysis of microbial communities. Overlaps of OTUs detected in (A) the three sample groups (Feces, ileum, ICLNs), and in (B) Feces-start and feces-end. Weighted UniFrac distances based on 16S rRNA gene libraries. Each point represents values from individual libraries with colors expressing (C) Ileum, Feces-start, Feces-end, and ileocaecal lymph node (ICLN) samples; (D) Control and antibiotic group samples (Feces-start samples were not separated into antibiotic-treated (AB) and control groups and are therefore not shown here); and (E) Pig samples. (F) OTU network of bacterial communities. Individual OTUs are represented by light yellow dots. Colored edges connect OTUs with the respective tissue in which they were detected: Ileum (red), Feces-start (blue), Feces-end (orange), ICLN (green). Colored nodes embody individual samples (same color code as edges). OTUs in the center of the network are shared among tissues, whereas OTUs on the outer rim are specifically present in certain tissues.

FIGURE 2

Taxonomic classification of 16S rRNA gene sequence reads. Taxonomic classification of 16S rRNA gene sequence reads parted by sampling site and group (AB or control). Data represents average of OTU counts from replicate libraries for each category. (A) Phylum-level classification. (B) Family-level classification. Phyla with less than 1% and families with less than 5% relative abundance (abund.) were grouped together. Sequences that could not be assigned are depicted as “Not assigned.” Start = Feces samples taken prior to antibiotic treatment (Thus not divided in AB and C group), AB = Antibiotic group, C = Control group, ICLN = Ileocecal lymph nodes.

FIGURE 3

Schematic drawing of the pig gastrointestinal (GI) tract anatomy. Pie charts represent mean relative abundances of 16S rRNA gene sequences associated to phylum level taxonomy. Percentage values in red depict proportion of OTUs that were significantly affected by AB treatment.

FIGURE 4

Relative abundance of 16S rRNA gene sequences associated to Candidatus Arthromitus. Boxes indicate the interquartile range (75th to 25th) of the data. The median value is shown as a line within the box. Whiskers extend to the most extreme value within 1.5 × interquartile range. Significance code: ^∗p = 0.0479.

FIGURE 5

Comparison of the microbial community composition in ICLNs from pig #1. (A) Relative abundances of 16S rRNA gene sequences. (B) Relative abundance of metatranscriptome reads. Genera with less than 1% relative abundance (abund.) were grouped together.