Bacterial Community Succession, Transmigration, and Differential Gene Transcription in a Controlled Vertebrate Decomposition Model

Abstract

Decomposing remains are a nutrient-rich ecosystem undergoing constant change due to cell breakdown and abiotic fluxes, such as pH level and oxygen availability. These environmental fluxes affect bacterial communities who respond in a predictive manner associated with the time since organismal death, or the postmortem interval (PMI). Profiles of microbial taxonomic turnover and transmigration are currently being studied in decomposition ecology, and in the field of forensic microbiology as indicators of the PMI. We monitored bacterial community structural and functional changes taking place during decomposition of the intestines, bone marrow, lungs, and heart in a highly controlled murine model. We found that organs presumed to be sterile during life are colonized by Clostridium during later decomposition as the fluids from internal organs begin to emulsify within the body cavity. During colonization of previously sterile sites, gene transcripts for multiple metabolism pathways were highly abundant, while transcripts associated with stress response and dormancy increased as decomposition progressed. We found our model strengthens known bacterial taxonomic succession data after host death. This study is one of the first to provide data of expressed bacterial community genes, alongside transmigration and structural changes of microbial species during laboratory controlled vertebrate decomposition. This is an important dataset for studying the effects of the environment on bacterial communities in an effort to determine which bacterial species and which bacterial functional pathways, such as amino acid metabolism, provide key changes during stages of decomposition that relate to the PMI. Finding unique PMI species or functions can be useful for determining time since death in forensic investigations.

Keywords: decomposition; metagenomic; metatranscriptomic; necrobiome; postmortem microbiome.

FIGURE 1

Experimental flow chart. A visual representation of the experimental design and downstream analyses. Mouse groups are represented as SSC = surface sterilized, colonized; NSSC = nonsurface sterilized, colonized; and CON = control.

FIGURE 2

Mouse decomposition stages. Images of mice before dissection and organ harvest for their selected postmortem times to represent the decomposition stages present at each timepoint.

FIGURE 3

S. aureus KUB7 detection with qPCR in organs as decomposition progresses. The log genomic units are plotted on the y-axis and postmortem time on the x-axis. Each circle represents a sample and the line represents the mean (±SE) log genomic units of S. aureus KUB7 during decomposition in the (A) lungs, (B) intestines, (C) heart, and (D) bone marrow.

FIGURE 4

Bacterial genera relative abundance in samples at postmortem times. The bacteria genera detected in (A) lungs, (B) intestines, (C) heart, and (D) bone marrow samples are represented by their relative percent abundance on the x-axis with the sample on the y-axis. The samples are labeled by postmortem timepoint group with the color bars [green = early (1 h, 3 h, 5 h), purple = middle (24 h), and yellow = late (7 d)]. Genera that constituted less than 3% of sample were grouped as rare taxa to reduce sampling noise. Relative abundances were determined using MetaPhlAn v2.0.

FIGURE 5

Distance-based RDA plots of the mouse organ microbiome over the postmortem interval. Distance-based RDA plots were created for both (A) Bray–Curtis and (B) Jaccard distances. Colors represent the postmortem intervals and shapes represent the organ from where the sample was obtained. Linear vectors were determined by genera with a significant environmental fit to the plot based on a p < 0.05.

FIGURE 6

Heatmaps of the significant pathway regulation during timepoint group comparisons. Heatmaps for (A) heart and (B) bone marrow representing the transcript count of each pathway annotated from the significant transcripts of each comparison by color. Pathway is included on the x-axis and timepoint group (EvL = early vs. late, MvL = middle vs. late) on the y-axis. Down-regulated transcripts were considered negatives and up-regulated transcripts were considered positives. Pathways that were not detected in a comparison are in gray.