Sheree J. Finley

Human Thanatomicrobiome Succession and Time Since Death

The thanatomicrobiome (thanatos, Greek for death) is a relatively new term and is the study of the microbes colonizing the internal organs and orifices after death. Recent scientific breakthroughs in an initial study of the thanatomicrobiome have revealed that a majority of the microbes within the human body are the obligate anaerobes, Clostridium spp., in the internal postmortem microbial communities. We hypothesized that time-dependent changes in the thanatomicrobiome within internal organs can estimate the time of death as a human body decays. Here we report a cross-sectional study of the sampling of 27 human corpses from criminal cases with postmortem intervals between 3.5–240 hours. The impetus for examining microbial communities in different internal organs is to address the paucity of empirical data on thanatomicrobiomic succession caused by the limited access to these organs prior to death and a dearth of knowledge regarding the movement of microbes within remains. Our sequencing results of 16S rRNA gene amplicons of 27 postmortem samples from cadavers demonstrated statistically significant time-, organ-, and sex-dependent changes. These results suggest that comprehensive knowledge of the number and abundance of each organ’s signature microorganisms could be useful to forensic microbiologists as a new source of data for estimating postmortem interval.

The Thanatomicrobiome: A Missing Piece of the Microbial Puzzle of Death.

Death is a universal phenomenon; however, is there “life after death?” This topic has been investigated for centuries but still there are gray areas that have yet to be elucidated. Forensic microbiologists are developing new applications to investigate the dynamic and coordinated changes in microbial activity that occur when a human host dies. There is currently a paucity of explorations of the thanatomicrobiome (thanatos-, Greek for death) and epinecrotic communities (microbial communities residing in and/or moving on the surface of decomposing remains). Ongoing studies can help clarify the structure and function of these postmortem microbiomes. Human microbiome studies have revealed that 75–90% of cells in the body prior to death are microbial. Upon death, putrefaction occurs and is a complicated process encompassing chemical degradation and autolysis of cells. Decomposition also involves the release of contents of the intestines due to enzymes under the effects of abiotic and biotic factors. These factors likely have predictable effects on postmortem microbial communities and can be leveraged for forensic studies. This mini review provides a critical examination of emerging research relating to thanatomicrobiome and epinecrotic communities, how each is studied, and possible strategies of stochastic processes.

Postmortem microbial communities in burial soil layers of skeletonized humans

Microorganisms are major ecological participants in the successional decomposition of vertebrates. The relative abundance, or the scarcity, of certain microbial taxa in gravesoil has the potential to determine the ecological status of skeletons. However, there are substantial knowledge gaps that warrant consideration in the context of the surrounding terrestrial ecosystem. In the current study, we hypothesized that i.) soil microbial diversity is disparate in the latter stage of decomposition (skeletonization) compared to the earlier stages (fresh, bloat, active and advanced decay), and ii.) the three layers of gravesoil (top, middle, and bottom) encompass similar microbial taxa and are analogous with control soil. To test these hypotheses, microbial communities in layers of burial soil of skeletonized bodies (treated) and from control soil, obtained from burial plots with no bodies (untreated), were compared using sequencing data of the 16S rRNA gene. The results demonstrated that Acidobacteria was confirmed as the most abundant microbial genus in all treated and untreated soil layers. Furthermore, Proteobacteria demonstrated a relatively low abundance in skeletonized gravesoil which is dissimilar from previous findings that assessed soil from earlier stages of human decomposition. Also, these results determined that soil microbial signatures were analogous in all three soil layers under the effects of similar abiotic and biotic factors, and they were similar to the communities in untreated soil. Therefore, the current study produced empirical data that give conclusive evidence of soil microbial successional changes, particularly for Proteobacteria, for potential use in forensic microbiology research.

Cadaver Thanatomicrobiome Signatures: The Ubiquitous Nature of Clostridium Species in Human Decomposition

Human thanatomicrobiome studies have established that an abundant number of putrefactive bacteria within internal organs of decaying bodies are obligate anaerobes, Clostridium spp. These microorganisms have been implicated as etiological agents in potentially life-threatening infections; notwithstanding, the scale and trajectory of these microbes after death have not been elucidated. We performed phylogenetic surveys of thanatomicrobiome signatures of cadavers’ internal organs to compare the microbial diversity between the 16S rRNA gene V4 hypervariable region and V3-4 conjoined regions from livers and spleens of 45 cadavers undergoing forensic microbiological studies. Phylogenetic analyses of 16S rRNA gene sequences revealed that the V4 region had a significantly higher mean Chao1 richness within the total microbiome data. Permutational multivariate analysis of variance statistical tests, based on unweighted UniFrac distances, demonstrated that taxa compositions were significantly different between V4 and V3-4 hypervariable regions (p < 0.001). Of note, we present the first study, using the largest cohort of criminal cases to date, that two hypervariable regions show discriminatory power for human postmortem microbial diversity. In conclusion, here we propose the impact of hypervariable region selection for the 16S rRNA gene in differentiating thanatomicrobiomic profiles to provide empirical data to explain a unique concept, the Postmortem Clostridium Effect.

Assessment of microbial DNA extraction methods of cadaver soil samples for criminal investigations

Gravesoil beneath decomposing cadavers undergoes substantial biochemical changes that have the potential to aid in PMI estimation and identification of clandestine gravesites. The use of DNA extraction methods is necessary for culture-independent downstream molecular applications such as PCR and next-generation sequencing. In this study, a comparison of four methods was performed for cadaver-soil collected beneath the heads and feet of 11 cadavers decaying in a natural setting. The four methods isolated 3.6–263 ng/μl of genomic DNA as determined by optical density analysis. The purity of the extracted DNA according to A260/280 and A260/230 ratios was determined. The A260/280 and A260/230 ratios were 1.24–1.97 and 0.27–2.12, respectively. The optical density at 320 nm was measured for humic acid quantification of the lysates from the method that provided the most efficient removal of humic acid. The results demonstrated that this method provided a 98% reduction of humic acid. PCR of 16S rRNA genes followed by gel electrophoresis was performed. The statistical analysis revealed a significant correlation between the yields and days on/in the soil using a phenol-chloroform method for soil collected at the head and feet. No earlier published work has extensively elucidated the efficacy of DNA extraction methods for DNA obtained from cadaver-soil.

Progression of thanatophagy in cadaver brain and heart tissues

Autophagy is an evolutionarily conserved catabolic process for maintaining cellular homeostasis during both normal and stress conditions. Metabolic reprogramming in tissues of dead bodies is inevitable due to chronic ischemia and nutrient deprivation, which are well-known features that stimulate autophagy. Currently, it is not fully elucidated whether postmortem autophagy, also known as thanatophagy, occurs in dead bodies is a function of the time of death. In this study, we tested the hypothesis that thanatophagy would increase in proportion to time elapsed since death for tissues collected from cadavers. Brain and heart tissue from corpses at different time intervals after death were analyzed by Western blot. Densitometry analysis demonstrated that thanatophagy occurred in a manner that was dependent on the time of death. The autophagy-associated proteins, LC3 II, p62, Beclin-1 and Atg7, increased in a time-dependent manner in heart tissues. A potent inducer of autophagy, BNIP3, decreased in the heart tissues as time of death increased, whereas the protein levels increased in brain tissues. However, there was no expression of BNIP3 at extended postmortem intervals in both brain and heart samples. Collectively, the present study demonstrates for the first time that thanatophagy occurs in brain and heart tissues of cadavers in a time-dependent manner. Further, our data suggest that cerebral thanatophagy may occur in a Beclin-1- independent manner. This unprecedented study provides potential insight into thanatophagy as a novel method for the estimation of the time of death in criminal investigationsAbstract: Autophagy is an evolutionarily conserved catabolic process for maintaining cellular homeostasis during both normal and stress conditions. Metabolic reprogramming in tissues of dead bodies is inevitable due to chronic ischemia and nutrient deprivation, which are well-known features that stimulate autophagy. Currently, it is not fully elucidated whether postmortem autophagy, also known as thanatophagy, occurs in dead bodies is a function of the time of death. In this study, we tested the hypothesis that thanatophagy would increase in proportion to time elapsed since death for tissues collected from cadavers. Brain and heart tissue from corpses at different time intervals after death were analyzed by Western blot. Densitometry analysis demonstrated that thanatophagy occurred in a manner that was dependent on the time of death. The autophagy-associated proteins, LC3 II, p62, Beclin-1 and Atg7, increased in a time-dependent manner in heart tissues. A potent inducer of autophagy, BNIP3, decreased in the heart tissues as time of death increased, whereas the protein levels increased in brain tissues. However, there was no expression of BNIP3 at extended postmortem intervals in both brain and heart samples. Collectively, the present study demonstrates for the first time that thanatophagy occurs in brain and heart tissues of cadavers in a time-dependent manner. Further, our data suggest that cerebral thanatophagy may occur in a Beclin-1- independent manner. This unprecedented study provides potential insight into thanatophagy as a novel method for the estimation of the time of death in criminal investigations

What Is the “Thanatomicrobiome” and What Is Its Relevance to Forensic Investigations?

Abstract Is death the end of life? In some ways it is; but in regards to the thanatomicrobiome (microbiome of death) on and in human remains, there are multitudes of living microorganisms after human death that have the potential to assist in medicolegal investigations. Determination of the precise cause and time since death, or postmortem interval, are crucial data for forensic science when criminal deaths are not witnessed, or when conflicting accounts are reported. Over the past decade, advances in high-throughput sequencing and bioinformatics have allowed the phylogenetic inventory of immense numbers of microorganisms in a variety of forensic applications. For example, several studies involve identifying humans using skin microbiota, determining body sample tissue sources, estimating postmortem interval, and identifying microorganisms. This chapter will discuss the current and emerging thanatomicrobiome technologies that forensically exploit microorganisms for use in criminal investigations and adjudication.

Erythroblast macrophage protein (Emp): Past, Present and Future

This review is a journey of the landmark erythroblast macrophage protein (Emp) discovered in 1994, and it walks chronologically through the progress that has been made in understanding the biological function of this protein. Historically, Emp was the first identified cell attachment molecule and is expressed in both erythroblasts and macrophages and mediates their attachments to form erythroblastic islands. The absence of Emp erythroblasts shows defects in differentiation and enucleation. Emp‐deficient macrophages display immature morphology characterized by small sizes, round shapes, and the lack of cytoplasmic projections. Although the primary sequence of Emp has already been determined and its role in both erythroid and macrophage development is well established, there are major gaps in the understanding of its function at the molecular level. Recent studies had implicated its importance in actin cytoskeleton remodeling and cell migration, but the molecular mechanisms are still enigmatic. Previous studies have also demonstrated that downregulation of Emp affects the expression of mitogen‐associated protein kinase 1 (MAPK1) and thymoma viral protooncogene (AKT‐1) resulting in abnormal cell motility. In this review, we summarize the proposed function of Emp based on previous studies, present scenarios, and its plausible future in translational research.