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The bones of decomposing vertebrates are colonized by a succession of diverse microbial communities. If this succession is similar across individuals, microbes may provide clues about the postmortem interval (PMI) during forensic investigations in which human skeletal remains are discovered. Here, we characterize the human bone microbial decomposer community to determine whether microbial succession is a marker for PMI. Six human donor subjects were placed outdoors to decompose on the soil surface at the Southeast Texas Applied Forensic Science facility. To also assess the effect of seasons, three decedents were placed each in the spring and summer. Once ribs were exposed through natural decomposition, a rib was collected from each body for eight time points at 3 weeks apart. We discovered a core bone decomposer microbiome dominated by taxa in the phylum Proteobacteria and evidence that these bone-invading microbes are likely sourced from the surrounding decomposition environment, including skin of the cadaver and soils. Additionally, we found significant overall differences in bone microbial community composition between seasons. Finally, we used the microbial community data to develop random forest models that predict PMI with an accuracy of approximately ±34 days over a 1- to 9-month time frame of decomposition. Typically, anthropologists provide PMI estimates based on qualitative information, giving PMI errors ranging from several months to years. Previous work has focused on only the characterization of the bone microbiome decomposer community, and this is the first known data-driven, quantitative PMI estimate of terrestrially decomposed human skeletal remains using microbial abundance information. IMPORTANCE Microbes are known to facilitate vertebrate decomposition, and they can do so in a repeatable, predictable manner. The succession of microbes in the skin and associated soil can be used to predict time since death during the first few weeks of decomposition. However, when remains are discovered after months or years, often the only evidence are skeletal remains. To determine if microbial succession in bone would be useful for estimating time since death after several months, human subjects were placed to decompose in the spring and summer seasons. Ribs were collected after 1 to 9 months of decomposition, and the bone microbial communities were characterized. Analysis revealed a core bone decomposer microbial community with some differences in microbial assembly occurring between seasons. These data provided time since death estimates of approximately ±34 days over 9 months. This may provide forensic investigators with a tool for estimating time since death of skeletal remains, for which there are few current methods.

Our ability to identify skeletal remains often relies on the quality and quantity of DNA extracted from bone and teeth. Current research on buried remains has been retrospective, and no study to our knowledge has comprehensively assessed both intra-individual and inter-individual variation in human skeletal DNA from all representative skeletal element types recovered from a burial. Three individuals were interred together in a single grave for four years. Following disinterment, skeletal DNA was extracted, quantified, and GlobalFiler™ results were produced from 49 bones per skeleton, representing all bone types. Multiple sites per bone were also tested to determine intra-bone variability. Co-extracted bacterial and fungal DNA were quantified to determine microbial loads in the bones. Results show that the small, cancellous bones of the feet outperformed other bones in terms of DNA yield, measured as nanograms per gram of bone powder, and short tandem repeat (STR) profile completeness. The cuneiforms, in particular, had consistently high human DNA yields for all three individuals. DNA yield varied by individual and depth within the grave, with the shallowest individual demonstrating the highest DNA yields While the feet exhibited the greatest variation in DNA yield across bone type and sampling site, they also demonstrated some of the highest DNA yields and the most complete STR profiles, evoking a re-evaluation of their use for skeletal DNA sampling and analysis.

Microbial colonization of bone is an important mechanism of postmortem skeletal degradation. However, the types and distributions of bone and tooth colonizing microbes are not well characterized. It is unknown if microbial communities vary in abundance or composition between bone element types, which could help explain differences in human DNA preservation. The goals of the present study were to (1) identify the types of microbes capable of colonizing different human bone types and (2) relate microbial abundances, diversity, and community composition to bone type and human DNA preservation. DNA extracts from 165 bone and tooth samples from three skeletonized individuals were assessed for bacterial loading and microbial community composition and structure. Random forest models were applied to predict operational taxonomic units (OTUs) associated with human DNA concentration. Dominant bacterial bone colonizers were from the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Planctomycetes. Eukaryotic bone colonizers were from Ascomycota, Apicomplexa, Annelida, Basidiomycota, and Ciliophora. Bacterial loading was not a significant predictor of human DNA concentration in two out of three individuals. Random forest models were minimally successful in identifying microbes related to human DNA concentration, which were complicated by high variability in community structure between individuals and body regions. This work expands on our understanding of the types of microbes capable of colonizing the postmortem human skeleton and potentially contributing to human skeletal DNA degradation.

Decomposing vertebrates, including humans, result in pronounced changes in surrounding soil biogeochemistry, particularly nitrogen (N) and carbon (C) availability, and alter soil micro- and macrofauna. However, the impacts of subsurface human decomposition, where oxygen becomes limited and microbial biomass is generally lower, are far less understood. The goals of this study were to evaluate the impact of human decomposition in a multi-individual, shallow (~70 cm depth) grave on soil biogeochemistry and soil microbial and nematode communities. Three individuals were interred and allowed to decay for four years. Soils were collected from two depths (0‒5 and 30‒35 cm) along linear transects radiating from the grave as well as from within and below (85‒90 cm depth) the grave during excavation to assess how decomposition affects soil properties. Along radiating surface transects, several extracellular enzymes rates and nematode richness increased with increasing distance from the grave, and likely reflect physical site disruption due to grave excavation and infill. There was no evidence of carcass-sourced C and N lateral migration from the grave, at least at 30‒35 cm depth. Within the grave, soils exhibited significant N-enrichment (e.g., ammonium, dissolved organic N), elevated electrical conductivity, and elevated respiration rates with depth. Soil biogeochemistry within the grave, particularly in the middle (30‒35 cm) and base (70‒75 cm depth), was significantly altered by human decomposition. Mean microbial gene abundances changed with depth in the grave, demonstrating increased microbial presence in response to ongoing decomposition. Human-associated Bacteroides were only detected at the base of the grave where anoxic conditions prevailed. Nematode community abundance and richness were reduced at 70‒75 cm and not detectable below 85‒90 cm. Further, we identified certain Plectus spp. as potential indicators of enrichment due to decomposition. Here we demonstrate that human decomposition influences soil biogeochemistry, microbes, and microfauna up to four years after burial.

Though recent decades have seen a marked increase in research concerning the impact of human decomposition on the grave soil environment, the fate of human DNA in grave soil has been relatively understudied. With the purpose of supplementing the growing body of literature in forensic soil taphonomy, this study assessed the relative persistence of human DNA in soil over the course of decomposition. Endpoint PCR was used to assess the presence or absence of human nuclear and mitochondrial DNA, while qPCR was used to evaluate the quantity of human DNA recovered from the soil beneath four cadavers at the University of Tennessee's Anthropology Research Facility (ARF). Human nuclear DNA from the soil was largely unrecoverable, while human mitochondrial DNA was detectable in the soil throughout all decomposition stages. Mitochondrial DNA copy abundances were not significantly different between decomposition stages and were not significantly correlated to soil edaphic parameters tested. There was, however, a significant positive correlation between mitochondrial DNA copy abundances and the human associated bacteria, Bacteroides, as estimated by 16S rRNA gene abundances. These results show that human mitochondrial DNA can persist in grave soil and be consistently detected throughout decomposition.