From Gut to Blood: Spatial and Temporal Pathobiome Dynamics during Acute Abdominal Murine Sepsis

Abstract

Abdominal sepsis triggers the transition of microorganisms from the gut to the peritoneum and bloodstream. Unfortunately, there is a limitation of methods and biomarkers to reliably study the emergence of pathobiomes and to monitor their respective dynamics. Three-month-old CD-1 female mice underwent cecal ligation and puncture (CLP) to induce abdominal sepsis. Serial and terminal endpoint specimens were collected for fecal, peritoneal lavage, and blood samples within 72 h. Microbial species compositions were determined by NGS of (cell-free) DNA and confirmed by microbiological cultivation. As a result, CLP induced rapid and early changes of gut microbial communities, with a transition of pathogenic species into the peritoneum and blood detected at 24 h post-CLP. NGS was able to identify pathogenic species in a time course-dependent manner in individual mice using cfDNA from as few as 30 microliters of blood. Absolute levels of cfDNA from pathogens changed rapidly during acute sepsis, demonstrating its short half-life. Pathogenic species and genera in CLP mice significantly overlapped with pathobiomes from septic patients. The study demonstrated that pathobiomes serve as reservoirs following CLP for the transition of pathogens into the bloodstream. Due to its short half-life, cfDNA can serve as a precise biomarker for pathogen identification in blood.

Keywords: CLP; NGS; cecal ligation puncture; cell-free DNA; gut microbiome; next-generation sequencing; pathobiome; pathogen liquid biopsy; sepsis.

Figure 1

Schematic illustration of the study design (A) and of the analytical workflow (B). (A): Three-month-old female mice (n = 45) were subjected to polymicrobial cecal ligation and puncture (CLP) sepsis (lower axis) or served as sham controls (upper axis) and were sampled in defined intervals for blood, feces, and abdominal lavage until 72 h post-CLP. Small blood drop indicates a serial small-volume sampling; large blood drop indicates terminal blood sampling. Short-interval serial blood sampling was performed maximally three times per individual mouse (i.e., serial blood sampling time points varied among mice to cover intervals smaller than 24 h) followed by terminal large-volume sampling. In each mouse, feces were collected at CLP and at the sacrifice time point. In each mouse, abdominal lavage was performed at the sacrifice time point. Body weight and temperature were taken daily. (B): NGS workflow was performed for all sample types. DNA was isolated, and all libraries were used for whole genome shotgun sequencing; nonmurine reads were classified and used for identification of species. Abdominal lavage and terminal blood were used for microbiological culturing in parallel. Growing cultures were used for species identification by means of MALDI TOF.

Figure 2

Healthy and post-CLP gut microbiome. (A): Healthy gut microbiome (0 h), percentage share of total normalized sequencing reads of top species shown for different mice. (B): Septic gut microbiome at 0 h, 24 h, and 48 h post-CLP, percentage share of total normalized sequencing reads of top species shown for different mice. (C): PCA of microbiomes at 0 h, 24 h, and 48 h post-CLP. Time points are indicated by color and shape of the symbols; bold symbols are centroids of the corresponding group.

Figure 3

Transition of microbes into peritoneum and blood as revealed by NGS and microbiology. (A–D): The most frequent bacterial species found in the lavage (A,C) and blood (B,D) detected by the culture-based methods (A,B) and by whole genome shotgun sequencing (C,D).

Figure 4

Overlap of microbes in the peritoneum and blood as revealed by NGS and microbiology. The most abundant species are shown for 0–72 h in sham and CLP mice. Heat maps (A,C) and Venn diagrams (B,D) of the most abundant species in the blood (A,B) and abdominal lavage (C,D) over 0–72 h in sham and CLP mice. Light blue squares, species detected by NGS (with >10 normalized reads); dark blue squares, species detected by NGS and either aerobic or anaerobic culture; gray squares, species detected by aerobic and/or anaerobic culture only.

Figure 5

Transitions of species from the feces to bloodstream/peritoneum at 24 h post-CLP in three mice. (A–C): Percentage of the most abundant species in feces at 0 h and 24 h, lavage at 24 h, and terminal blood sample at 24 h after CLP. (D): Community diversity and richness shown for all sample types of mouse 3 indicated by Shannon–Wiener diversity index and ACE richness index. Standard errors are shown.

Figure 6

Abundance of the most prominent genera in the blood. Blood samples collected from septic humans (n = 239 samples of 48 individuals at different time points) and mice (n = 49 terminal blood samples of 49 individuals). The seven most prominent genera found in humans and mice are shown; their abundance is indicated by the percentage of blood samples showing >10 normalized sequencing reads for the respective genus out of all available blood samples.

Figure 7

(A): Concentration for top species shown in molecules/mL of serial blood samples for two mice at different time points. The numbers above bars indicate the number of detected top species. Crosses indicate a corresponding IL-6 concentration in the blood per time point. (B–D): Time course of the most abundant species in molecules/mL of serial blood samples in two individuals.