Bridging the gap between decomposition theory and forensic research on postmortem interval.

Knowledge of the decomposition of vertebrate animals has advanced considerably in recent years and revealed complex interactions among biological and environmental factors that affect rates of decay. Yet this complexity remains to be fully incorporated into research or models of the postmortem interval (PMI). We suggest there is both opportunity and a need to use recent advances in decomposition theory to guide forensic research and its applications to understanding the PMI. Here we synthesise knowledge of the biological and environmental factors driving variation in decomposition and the acknowledged limitations among current models of the PMI. To guide improvement in this area, we introduce a conceptual framework that highlights the multiple interdependencies affecting decay rates throughout the decomposition process. Our framework reinforces the need for a multidisciplinary approach to PMI research, and calls for an adaptive research cycle that aims to reduce uncertainty in PMI estimates via experimentation, modelling, and validation.

Human Bone Proteomes before and after Decomposition: Investigating the Effects of Biological Variation and Taphonomic Alteration on Bone Protein Profiles and the Implications for Forensic Proteomics.

Bone proteomic studies using animal proxies and skeletonized human remains have delivered encouraging results in the search for potential biomarkers for precise and accurate post-mortem interval (PMI) and the age-at-death (AAD) estimation in medico-legal investigations. The development of forensic proteomics for PMI and AAD estimation is in critical need of research on human remains throughout decomposition, as currently the effects of both inter-individual biological differences and taphonomic alteration on the survival of human bone protein profiles are unclear. This study investigated the human bone proteome in four human body donors studied throughout decomposition outdoors. The effects of ageing phenomena (in vivo and post-mortem) and intrinsic and extrinsic variables on the variety and abundancy of the bone proteome were assessed. Results indicate that taphonomic and biological variables play a significant role in the survival of proteins in bone. Our findings suggest that inter-individual and inter-skeletal differences in bone mineral density (BMD) are important variables affecting the survival of proteins. Specific proteins survive better within the mineral matrix due to their mineral-binding properties. The mineral matrix likely also protects these proteins by restricting the movement of decomposer microbes. New potential biomarkers for PMI estimation and AAD estimation were identified. Future development of forensic bone proteomics should include standard measurement of BMD and target a combination of different biomarkers.

New evidence of predation on humans by cookiecutter sharks in Kauai, Hawaii.

The feeding patterns of species of large sharks on human corpses are well documented in the literature however, that of smaller sharks are less known. This may introduce uncertainty in the medicolegal conclusions. For that reason, accurate identification of patterns of shark predation is very relevant, specifically in areas bordered by the sea. In the case described here, an unidentified lesion was noted on the body of a victim of a scuba diving accident off the island of Kauai, in Hawaii. The aim of this study was to identify the origin of the lesion and investigate its potential to inform on the context of death and/or decomposition. The original outline of the lesion was digitally reconstructed to enable the collection of measurements which were compared with the literature and interpreted with an interdisciplinary approach. This approach permitted to determine that the macroscopic appearance and dimensions of the lesion (major axis = 3.53 cm) were consistent with a bitemark of a cookiecutter shark (Isistius brasiliensis). It was further determined that the bitemark was incomplete and that the specimen involved had a total length of about 24 cm and was likely to be a juvenile. This is the second report in the published literature of cookiecutter bitemarks on humans in the Hawaiian waters. This study brings new evidence-based insights into the interactions between cookiecutter sharks and human remains in marine environments and provides a valuable contribution to the knowledge base on the topic.

The suitability of visual taphonomic methods for digital photographs: An experimental approach with pig carcasses in a tropical climate.

In the context of increased scrutiny of the methods in forensic sciences, it is essential to ensure that the approaches used in forensic taphonomy to measure decomposition and estimate the postmortem interval are underpinned by robust evidence-based data. Digital photographs are an important source of documentation in forensic taphonomic investigations but the suitability of the current approaches for photographs, rather than real-time remains, is poorly studied which can undermine accurate forensic conclusions. The present study aimed to investigate the suitability of 2D colour digital photographs for evaluating decomposition of exposed human analogues (Sus scrofa domesticus) in a tropical savanna environment (Hawaii), using two published scoring methods; Megyesi et al., 2005 and Keough et al., 2017. It was found that there were significant differences between the real-time and photograph decomposition scores when the Megyesi et al. method was used. However, the Keough et al. method applied to photographs reflected real-time decomposition more closely and thus appears more suitable to evaluate pig decomposition from 2D photographs. The findings indicate that the type of scoring method used has a significant impact on the ability to accurately evaluate the decomposition of exposed pig carcasses from photographs. It was further identified that photographic taphonomic analysis can reach high inter-observer reproducibility. These novel findings are of significant importance for the forensic sciences as they highlight the potential for high quality photograph coverage to provide useful complementary information for the forensic taphonomic investigation. New recommendations to develop robust transparent approaches adapted to photographs in forensic taphonomy are suggested based on these findings.

Carcass mass has little influence on the structure of gravesoil microbial communities.

Little is known about how variables, such as carcass mass, affect the succession pattern of microbes in soils during decomposition. To investigate the effects of carcass mass on the soil microbial community, soils associated with swine (Sus scrofa domesticus) carcasses of four different masses were sampled until the 15th day of decomposition during the month of June in a pasture near Lincoln, Nebraska. Soils underneath swine of 1, 20, 40, and 50 kg masses were investigated in triplicate, as well as control sites not associated with a carcass. Soil microbial communities were characterized by sequencing the archaeal, bacterial (16S), and eukaryotic (18S) rRNA genes in soil samples. We conclude that time of decomposition was a significant influence on the microbial community, but carcass mass was not. The gravesoil associated with 1 kg mass carcasses differs most compared to the gravesoil associated with other carcass masses. We also identify the 15 most abundant bacterial and eukaryotic taxa, and discuss changes in their abundance as carcass decomposition progressed. Finally, we show significant decreases in alpha diversity for carcasses of differing mass in pre-carcass rupture (days 0, 1, 2, 4, 5, and 6 postmortem) versus post-carcass rupture (days 9 and 15 postmortem) microbial communities.

An initial investigation into the ecology of culturable aerobic postmortem bacteria.

Postmortem microorganisms are increasingly recognized for their potential to serve as physical evidence. Yet, we still understand little about the ecology of postmortem microbes, particularly those associated with the skin and larval masses. We conducted an experiment to characterize microbiological and chemical properties of decomposing swine (Sus scrofa domesticus) carcasses on the island of Oahu, Hawaii, USA, during June 2013. Bacteria were collected from the head, limb, and larval mass during the initial 145h of decomposition. We also measured the pH, temperature, and oxidation-reduction potential of larval masses in situ. Bacteria were cultured aerobically on Standard Nutrient Agar at 22°C and identified using protein or genetic signals. Carcass decomposition followed a typical sigmoidal pattern and associated bacterial communities differed by sampling location and time since death, although all communities were dominated by phyla Actinobacteria, Firmicutes, and Proteobacteria. Larval masses were reducing environments (~-200mV) of neutral pH (6.5-7.5) and high temperature (35°C-40°C). We recommend that culturable postmortem and larval mass microbiology and chemistry be investigated in more detail, as it has potential to complement culture-independent studies and serve as a rapid estimate of PMI.

Seasonal variation of postmortem microbial communities.

Body-associated microbes were recently shown to change significantly during decomposition, undergoing an ecological succession in experimental conditions using rodent and swine models. We investigated microbial succession in soils associated with swine carcasses under experimental field conditions in summer and winter. We demonstrate that these postmortem microbial communities change in a specific, reproducible fashion, and that soil microbes represent a significant component of the postmortem microbial community, contrary to widespread belief in forensic science. However, the effects of decomposition on soil microbial communities were different in summer and winter. We suggest that the microbial ecological succession will be useful in medicolegal death investigation; however, observations in winter might not be applicable to summer, which indicates a need for a greater understanding of the seasonality of decomposition.

Vertebrate decomposition is accelerated by soil microbes.

Carrion decomposition is an ecologically important natural phenomenon influenced by a complex set of factors, including temperature, moisture, and the activity of microorganisms, invertebrates, and scavengers. The role of soil microbes as decomposers in this process is essential but not well understood and represents a knowledge gap in carrion ecology. To better define the role and sources of microbes in carrion decomposition, lab-reared mice were decomposed on either (i) soil with an intact microbial community or (ii) soil that was sterilized. We characterized the microbial community (16S rRNA gene for bacteria and archaea, and the 18S rRNA gene for fungi and microbial eukaryotes) for three body sites along with the underlying soil (i.e., gravesoils) at time intervals coinciding with visible changes in carrion morphology. Our results indicate that mice placed on soil with intact microbial communities reach advanced stages of decomposition 2 to 3 times faster than those placed on sterile soil. Microbial communities associated with skin and gravesoils of carrion in stages of active and advanced decay were significantly different between soil types (sterile versus untreated), suggesting that substrates on which carrion decompose may partially determine the microbial decomposer community. However, the source of the decomposer community (soil- versus carcass-associated microbes) was not clear in our data set, suggesting that greater sequencing depth needs to be employed to identify the origin of the decomposer communities in carrion decomposition. Overall, our data show that soil microbial communities have a significant impact on the rate at which carrion decomposes and have important implications for understanding carrion ecology.

Volatile organic compounds produced during postmortem processes can be linked via chromatographic profiles to individual postmortem bacterial species.

Decomposition odor is produced during postmortem mammalian tissue breakdown by bacteria, insects, and intrinsic chemical processes. Past research has not thoroughly investigated which volatile organic compounds (VOCs) can be linked directly to individual bacterial species on decomposing remains. The purpose of this study was to profile the VOCs produced over time by individual species of bacteria using comprehensive two-dimensional gas chromatography (GC×GC) to expand our foundational knowledge of what each bacterial species contributes to decomposition odor. Five different species of bacteria (Bacillus subtilis, Ignatzschineria indica, Ignatzschineria ureiclastica, Curtobacterium luteum, and Vagococcus lutrae) were cultured on standard nutrient agar individually and monitored daily using solid phase microextraction arrow (SPME Arrow) and GC×GC in combination with quadrupole mass spectrometry (qMS) and flame ionization detection (FID). The GC×GC-qMS/FID approach was used to generate rich VOC profiles that represented the bacterial species' metabolic VOC production longitudinally. The data obtained from the chromatographic output was used to compare with a prior study using one-dimensional GC-qMS, and also between each of the five species to investigate the extent of overlap between species. No single VOC could be found in all five bacterial species investigated, and there was little overlap in the profile between species. To further visualize these differences, chromatographic peak data was investigated using two different ordination strategies, principal component analysis (PCA) and principal coordinate analysis (PCoA). The two ordination strategies were compared with each other using a Procrustes analysis. This was performed to understand differences in ordination strategies between the separation science community and chemical ecological community. Overall, ordination strategies were found to produce similar results, as evidenced by the correlation of PCA and PCoA in the Procrustes analysis. All analysis strategies yielded distinct VOC profiles for each species. Further study of additional species will support understanding of the holistic view of decomposition odor from a chemical ecology perspective, and further support our understanding of the production of decomposition odor that culminates from such a complex environment.

A conserved interdomain microbial network underpins cadaver decomposition despite environmental variables.

Microbial breakdown of organic matter is one of the most important processes on Earth, yet the controls of decomposition are poorly understood. Here we track 36 terrestrial human cadavers in three locations and show that a phylogenetically distinct, interdomain microbial network assembles during decomposition despite selection effects of location, climate and season. We generated a metagenome-assembled genome library from cadaver-associated soils and integrated it with metabolomics data to identify links between taxonomy and function. This universal network of microbial decomposers is characterized by cross-feeding to metabolize labile decomposition products. The key bacterial and fungal decomposers are rare across non-decomposition environments and appear unique to the breakdown of terrestrial decaying flesh, including humans, swine, mice and cattle, with insects as likely important vectors for dispersal. The observed lockstep of microbial interactions further underlies a robust microbial forensic tool with the potential to aid predictions of the time since death.

Developing a new scoring method to evaluate human decomposition in a humid, continental (Dfb) climate in Quebec.

The published literature shows a lack of methods to evaluate the patterns and extent of decomposition of human remains and to estimate the post-mortem interval (PMI) in humid, continental (Dfb) climates such as Quebec. The aim of this study was to address this gap in the current knowledge base by providing the first observations from human corpses studied under controlled conditions in Quebec. A 12-month study was conducted at the site for Research in Experimental and Social Thanatology; the first human taphonomy facility in Canada. Six human donors with known time of death were deposited across spring (n = 1), summer (n = 3), and autumn (n = 2) 2021. The lack of suitability of the total body score method to evaluate the extent of decomposition at the facility prompted the development of a new scoring system based on the macromorphoscopic changes observed. The scoring system was applied to the donors to evaluate decomposition throughout seasons. All donors followed comparable decomposition trajectories, regardless of the season of deposition. Eighty-five percent of taphonomic patterns appeared in the first 25 experimental days or 5000 Kelvin accumulated degree days (350 ADD). Extensive desiccation of tissues was observed at a median of 21 experimental days across donors, resulting in a plateau within decomposition with no extensive skeletonization. To the authors' knowledge, this is the first published report of experimentally observed desiccation in such a form in a Dfb climate. This study provides new data on the types of decomposition patterns to expect in forensic investigations in southern Quebec and comparable climates.

A synthesis of carcass decomposition studies conducted at a tropical (Aw) taphonomy facility: 2013-2022.

Decomposition studies have been conducted in several regions of the world, but relatively few have investigated taphonomy in tropical environments. Even fewer have explored carcass decomposition during multiple tropical seasons, leaving the relationships between season and decomposition in tropical environments poorly understood. Ten decomposition studies using 30 carcasses were conducted in Honolulu, Hawaii, USA to start addressing this knowledge gap. These studies show that some postmortem processes were observed regardless of season. Carcass temperature and chemistry were spatiotemporally variable. Fly larval masses were consistently observed within 3 days (∼75 ADD) postmortem and carcasses lost 60%-90% of mass by 10 days (∼250 ADD) postmortem (Total Body Score ∼26). Season had a significant effect on decomposition, yet the warmest and most humid seasons did not always result in the most rapid and extensive decomposition. Seasonal variation appears to be less pronounced than at other tropical decomposition sites.

Postmortem Skeletal Microbial Community Composition and Function in Buried Human Remains.

Bones and teeth can provide a lasting resource to identify human remains following decomposition. Bone can support dynamic communities of micro- and macroscopic scavengers and incidental taxa, which influence the preservation of bone over time. Previously we identified key microbial taxa associated with survivability of DNA in bones of surface-decomposed human remains, observing high intra- and interindividual variation. Here we characterized the postmortem bone microbiome of skeletal remains in a multi-individual burial to better understand subsurface bone colonization and preservation. To understand microbial community origins and assembly, 16S rRNA amplicon sequences from 256 bone and 27 soil samples were compared to bone from individuals who decomposed on the ground surface, and human gut sequences from the American Gut Project. Untargeted metabolomics was applied to a subset of 41 bone samples from buried remains to examine potential microbe-metabolite interactions and infer differences related to community functionality. Results show that postmortem bone microbial communities are distinct from those of the oxic surface soils and the human gut. Microbial communities from surface-deposited bone and shallow buried bone were more similar to those from soils, while bones recovered from saturated areas deeper in the grave showed increased similarity with human gut samples with higher representation of anaerobic taxa, suggesting that the depositional environment affected the established bone microbiome. Correlations between metabolites and microbes indicate that phosphate solubilization is likely an important mechanism of microbially mediated skeletal degradation. This research expands our knowledge of microbial bone colonizers, including colonizers important in a burial environment. IMPORTANCE Understanding the microbes that colonize and degrade bone has important implications for preservation of skeletal elements and identification of unknown human remains. Current research on the postmortem bone microbiome is limited and largely focuses on archaeological or marine contexts. Our research expands our understanding of bone microbiomes in buried remains by characterizing the taxonomic and metabolic diversity of microbes that are colonizing bone after a 4-year postmortem burial interval and examines the potential impact of microbial colonization on human skeletal DNA preservation. Our results indicate that the postmortem bone microbiome is distinct from the human gut and soil. Evidence from combined metabolomic and amplicon sequencing analysis suggests that Pseudomonas and phosphate solubilization likely play a role in skeletal degradation. This work provides important insight into the types and activities of microbes controlling the preservation of buried skeletal remains.

A Pilot Study of Microbial Succession in Human Rib Skeletal Remains during Terrestrial Decomposition.

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.

Volatile Organic Compound Profiling from Postmortem Microbes using Gas Chromatography-Mass Spectrometry.

Volatile organic compounds (VOCs) are by-products of cadaveric decomposition and are responsible for the odor associated with decomposing remains. The direct link between VOC production and individual postmortem microbes has not been well characterized experimentally. The purpose of this study was to profile VOCs released from three postmortem bacterial isolates (Bacillus subtilis, Ignatzschineria indica, I. ureiclastica) using solid-phase microextraction arrow (SPME Arrow) and gas chromatography-mass spectrometry (GC-MS). Species were inoculated in headspace vials on Standard Nutrient Agar and monitored over 5 days at 24°C. Each species exhibited a different VOC profile that included common decomposition VOCs. VOCs exhibited upward or downward temporal trends over time. Ignatzschineria indica produced a large amount of dimethyldisulfide. Other compounds of interest included alcohols, aldehydes, aromatics, and ketones. This provides foundational data to link decomposition odor with specific postmortem microbes to improve understanding of underlying mechanisms for decomposition VOC production.

Characterizing the postmortem human bone microbiome from surface-decomposed remains.

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.

The microbiology, pH, and oxidation reduction potential of larval masses in decomposing carcasses on Oahu, Hawaii.

Previous studies have begun to characterize the microbial community dynamics of the skin, soil, gut, and oral cavities of decomposing remains. One area that has yet to be explored in great detail is the microbiome of the fly larval mass, the community of immature flies that plays a significant role in decomposition. The current study aimed to characterize the microbiology and chemistry of larval masses established on pig (Sus scrofa domesticus) carcasses and to determine if these characteristics have potential as temporal evidence. Carcasses (n = 3) were decomposed on the soil surface of a tropical habitat on Oahu, Hawaii, USA and sampled over three days at 74 h, 80 h, 98 h, 104 h, 122 h, and 128 h (∼85-142 Accumulated Degree Days) postmortem. Larval masses were analyzed via high-throughput 16S rRNA sequencing and in situ chemical measurements (pH, temperature, oxidation-reduction potential). A trend was observed that resulted in three distinct microbial communities (pre-98 h, 98 h, and post-98 h). The oxidation-reduction potential (Eh) of larval masses apparently regulated microbial community structure with the most negative Eh being associated with the least rich and diverse microbial communities. Overall, a significant interaction between time and taxa was observed, particularly with bacterial phyla Firmicutes and Proteobacteria. The current results provide new insight into the microbial community and chemical parameters of larval masses and indicate a temporal shift that could be further studied as a PMI estimator.

An Experiment to Characterize the Decomposer Community Associated with Carcasses (Sus scrofa domesticus) on Oahu, Hawaii.

Pig (Sus scrofa domesticus) carcasses were decomposed on the soil surface of a terrestrial habitat on the island of Oahu, Hawaii to begin characterizing the decomposer community. Results showed that carcasses can decompose rapidly on Oahu, primarily due to the activity of fly larvae, with ~80% of mass lost by 8 days (~220 ADD) postmortem. Scavenging was conducted exclusively by the small Indian mongoose (Herpestes javanicus), first feeding on larvae then feeding on the remains. Carcasses were habitats of warm temperature, little to no oxygen, slightly acidic/neutral pH, and high sodium concentration. Larval masses selected for a microbial community comprised of multiple bacterial taxa from phyla Firmicutes and Proteobacteria, particularly genera Clostridium, Proteus, and Providencia. These larval masses were well established from 3 to 8 days (~90 to ~220 ADD) postmortem. These data provide helpful, novel insight into the structure and activity of carcass decomposer communities on Oahu.

Trace Evidence Potential in Postmortem Skin Microbiomes: From Death Scene to Morgue.

Microbes can be used effectively as trace evidence, at least in research settings. However, it is unknown whether skin microbiomes change prior to autopsy and, if so, whether these changes interfere with linking objects to decedents. The current study included microbiomes from 16 scenes of death in the City and County of Honolulu and tested whether objects at the scenes can be linked to individual decedents. Postmortem skin microbiomes were stable during repeated sampling up to 60 h postmortem and were similar to microbiomes of an antemortem population. Objects could be traced to decedents approximately 75% of the time, with smoking pipes and medical devices being especially accurate (100% match), house and car keys being poor (0%), and other objects like phones intermediate (~80%). These results show that microbes from objects at death scenes can be matched to individual decedents, opening up a new method of establishing associations and identifications.

Using ninhydrin to detect gravesoil.

Some death scene investigations commence without knowledge of the location of the body and/or decomposition site. In these cases, it is necessary to locate the remains or the site where the body decomposed prior to movement. We hypothesized that the burial of a mammalian cadaver will result in the release of ninhydrin reactive nitrogen (NRN) into associated soil and that this reaction might have potential as a tool for the identification of clandestine graves. Juvenile rat (Rattus rattus) cadavers were buried in three contrasting soil types in Australian tropical savanna ecosystems and allowed to decompose over a period of 28 days. Soils were sequentially harvested and analyzed for NRN. Cadaver burial resulted in an approximate doubling (mean = 1.7 +/- 0.1) in the concentration of soil NRN. This reaction has great potential to be used as a presumptive test for gravesoil and this use might be greatly enhanced following more detailed research.