Our findings characterize the spatial and temporal heterogeneity of the human postmortem microbiome across a large-scale study. By focusing on the microbiota after death, we highlight that ecological selection is the driving force of community assembly, and the potential to further expand the knowledge of human microbiomes across populations through utilization of an often-overlooked yet ubiquitously available resource – the postmortem microbiome. Analysis of the impact of host and environmental factors from diverse populations will likely provide insights that can promote testing postmortem microbial communities as indicators of human health and forensics. The evaluation of postmortem microbial consortia may transform our understanding of human health among large populations by supporting evidence that long-term surveillance of microbiomes at or within a day of death could have broad public health and forensic relevance.

Further, there are challenges for researchers studying microbial assemblages in complex and uncontrolled environmental conditions. One initial challenge that our group overcame was consistent and reliable sampling during routine death investigation processes. We did so by developing standardized protocols and training investigators and autopsy staff to systematically and consistently collect samples as part of their routine daily caseload. Establishing these methodologies is vital to expand the potential utility of postmortem microbial communities as a surveillance tool for human health or for use in forensics. Another is the potential for cross-contamination to occur during autopsy in the larger offices were multiple autopsies are often conducted simultaneously. However, our results demonstrate there are distinct community differences and succession patterns among all cases. If cross-contamination was of significant biological importance we would expect that microbial communities among cases would be more similar regardless of anatomic area, age, sex, ethnicity, weight status, estimated PMI, antemortem health conditions, or manner of death: we did not find evidence of cross-contamination.

Cross-Sectional Successional Patterns

Our results demonstrate that despite the fluidic process of decomposition and decay, the body maintains strong niche differentiation among anatomic locations. The umbilicus sample did not provide useful results for the purposes for this study, as indicated by analysis of the beta diversity (Table S2; Fig. S3), and the umbilicus only resulted in samples with adequate post-filtering sequences in half of all cases (50%). Further, we did not conclude there was a homogenization of microbial communities across all anatomic sites as the time since death increased (Figs S4 and S8), as others have noted distinct microbial communties based on anatomic location in living hosts1,7,26. However, we acknowledge the skewed sampling to cases with an estimated PMI of less than two days (87%). This is because, as a practical matter, most deaths are reported or discovered a short time after they happen regardless of the size of the community, the vast majority within 48 hours of death. Thus, our dataset is important for establishing a foundational baseline of postmortem microbial characterization that may be useful in future forensic applications because, as far as we know, mid-sized and larger death investigation systems in the United States have comparable practices so that our results may be applicable elsewhere. It is probable that the microbial communities of all anatomic locations become more similar to each other over prolonged periods of decomposition (e.g., weeks to months), or the body becomes more similar to the environmental microbial communities, such as soil or aquatic habitats, as found in other studies10,11,12,24,25,27,28.

The demographics of our population may be influencing the structure and function of the reported microbiome microbial communities. Our dataset is comprised of 52.1% of cases being white only, which reflects the 2016 US Census17, that determined the population of Wayne County (MI) is 54.6% (white only). It is important to note that the demographics of this dataset are reflective of the composition of the living population in Wayne County, and not a skewed subset of an a priori targeted or self-selecting demographic as is common of many living microbiome studies1,2,4,8,26,29,30,31. The goal of this initially large-scale survey was to identify the covariates most important in structuring microbial communities after death for a population not preselected based on health condition.

The relationship of the alpha diversity metrics with the age indicated a weak negative relationship with diversity metrics for all anatomic locations, except the ears. These results provide initial evidence that there is reduced richness, diversity and evenness in the postmortem microbiome with aging. The same relationships were tested with BMI instead of age, and a weak positive relationship emerged between BMI and the alpha diversity metrics across all anatomic locations. Previous surveys of the postmortem microbial communities for internal organs have also demonstrated sex was nearly significant (p = 0.05) in structuring microbial richness and not organ type, age, estimated PMI, or ambient temperature; however, organ type and sex had significant differences based on Shannon diversity32. Results from this dataset (Table S3, Figs S3, S5 and S6) demonstrated manner of death, sex, estimated postmortem interval, death event location, ethnicity, season of death, and body weight (in descending order of significance) were not the most important covariates structuring the microbial communities. It has been argued that ethnicity is a reflection of social and political groupings with little biological basis33. Microbial taxon differences have been observed in living individuals with differing self-reported results of race/ethnicity1,34, despite ethnicity not being a strong covariate (p > 0.6) structuring our dataset. The weight of an individual has been shown to be important in structuring microbial communities in living individuals1,5,35,36. However, the postmortem microbial communities could also be influenced by other factors, such as the built, urban environment, undisclosed health conditions, medications or other environmental chemicals, which were not explicitly tested using this dataset. Future in-depth analysis of the influence of specifics covariates on postmortem microbial communities is warranted.

To test the variability of taxa over decomposition time, we plotted the mean taxon abundance by their variability and determined within each anatomic area the relationships followed Taylor’s (power) law. Mathematically, Taylor’s law should have a slope of two37. But it is common within naturally occurring ecological systems to have a slope less than two38. A slope less than two indicates that species dominating populations should be less variable in space or time; this relationship of predominate species maintaining their population size could be caused by negative species interactions (direct or apparent competition) or interspecific competition38. For our dataset, the combination of increased slopes after two days since death with lower phylogenetic diversity after two days postmortem suggests that negative species interactions could be a potential mechanism of succession.

Further, we documented that most variability over decomposition occurred in the microbial communities of the mouth, while community membership and structure were most consistent in the rectum. These results suggest that the external communities are more subjected to physics and the environmental interactions while internal communities are more influenced by biochemical processes. Despite the dimensionality of our dataset, which may be explained by biotic interactions, stochastic effects, host genetics, or other unknown factors, the prevalence of common taxa and composition detected in the first two days postmortem suggests that microbial communities are not undergoing rapid turnover within 24–48 h of host death. Therefore, we postulate the taxa of the first 24–48 h after death will most represent antemortem microbial communities.

Comparisons to Previous Postmortem Human Microbiome Studies

Our large-scale survey of the human postmortem microbiome clearly demonstrates that microbial community composition changed among anatomic areas and over time. Notably, our study has approximately three times the number of cases than previous studies, and we report predictive in silico function profiles from a large-scale human postmortem database. Due to the large number of cases analyzed (188 cases), we were able to examine the observed power (Table S8), which has not been previously reported in other human postmortem microbiome studies12,13,24,25,32,39,40,41,42,43,44. Differences in the microbial communities less than two days compared to greater than two days after death gave us the most power (>0.8) to detect large effect sizes (d > 0.5) in the mouth and ears. The remaining anatomic locations (eyes, nose, rectum) had less power (<0.8) to detect differences in the microbial communities given the sample sizes (~150 cases < 48 h PMI vs. ~23 cases > 49 PMI). This dataset also has key differences in experimental design, sampling methodologies, and study aims compared to previous studies (see Table 1 for summary). For example, one study profiled bacterial signatures on bones39, which is beyond the current scope of this dataset. Other studies focused on surveying the postmortem microbial communities of internal organs and blood32,42,43, and thus a direct comparison cannot be made based the anatomic locations sampled for this dataset. We also recognize the time a body has been decomposing (either estimated postmortem interval or time since placement in the field at anthropological facilities) is different when comparing our dataset to previous work from anthropological facility10,12,13,24,25 or surveys of the internal organs during death investigation with a smaller sample size (n ≤ 46 cases)32,43.

Anatomic location influenced the microbial diversity, with the rectum and eyes having the most phylogenetic diversity while the ears, nose, and mouth having the least diversity (Fig. 1B). These data partially agree with previously reported diversity trends from two bodies that showed increased microbial richness as sampling moved from the upper to the lower the gastrointestinal tract12. Another study to survey the microbial communities of the liver, spleen, heart, brain, blood, and mouth of 28 bodies, detected increased richness in the mouth compared to the internal anatomic locations and blood samples43. Further, we observed a decrease in alpha diversity metrics (richness and phylogenetic diversity) as the estimated postmortem interval increased (greater than two days after death) in all anatomic locations sampled except the rectum (Fig. 1B). These results are consistent with some previously published data that documented a decrease in microbial diversity in the auditory canal from 21 bodies13; only four bodies in that study had repeated sampling events up to 800 accumulated degree days with the remaining 17 bodies having single sampling events to characterize the microbial communities. However, our data do not align with another study that characterized the microbial communities of two cadavers during decomposition in two seasons (spring and winter). Metcalf et al.10 did not detect statistical differences (alpha = 0.05) in the phylogenetic diversity of skin sites (e.g., hips, biceps, head, groin) over decomposition. Javan et al.32 also did not detect significant differences (alpha = 0.05) in Chao1 richness or Shannon diversity from samples collected from internal organs (liver, spleen, heart, brain, and blood) and the mouth in cases with estimated PMIs from 3.5–240 h; however, alpha diversity metrics of each sampling location were not presented. Thus, it is undetermined whether the postmortem microbial communities of the mouth were similar in diversity over decomposition time. Overall, the similarity of our dataset to previous studies is strongest when comparing microbial diversity based on topographical distribution. The postmortem microbial communities detected in the mouth were less diverse than those of the rectum. Further, the decline in alpha diversity as decomposition time increased could result from increased competition in the microbial communities later in decomposition (after two days of death) or a successional shift to a more anaerobic taxa configuration.

In each anatomic area, distinct compositional changes were observed at the phyletic level. Actinobacteria and Bacteroidetes abundance decreased after the first two days of decomposition, while Proteobacteria abundance increased after two days of decomposition (Fig. S7A,B). A specific taxon within Bacteroidetes (i.e., Prevotella) has been previously detected from mouth microbial communities during the earlier stages of decomposition in both anthropological field studies12 and death investigation cases43. Additionally, the decline in Bacteroidetes from this dataset is similar to decreased abundance of Bacterioides from 12 bodies at an anthropological facility25, despite microbial samples being characterized from the proximal large intestine. Hauther et al.25 performed a targeted study to characterize postmortem dynamics of three common gut taxa during decomposition (9 to 20 days), and also documented a decline in Lactobacillus abundance while Bifidobacterium did not change during decomposition. We also detected a lower abundance of Firmicutes, specifically Staphylococcus and Streptococcus, abundance after the first two days of decomposition in all anatomic locations except the nose (Figs 2B and S7A,B). These results are similar to previous studies that documented Streptococcus was a predominant taxon in the mouth: during pre-bloat for two bodies decomposing in an anthropological research facility12; during the first four days of decomposition for three bodies decomposition in an anthropological research facility44; and from 13 death investigation cases with estimated PMI of 10–70 h43. ANCOM tests also indicated Streptococcus as a potential biomarker in the eyes and mouth during the first two days after death. Additionally, Staphylococcus detected in the ear form 21 bodies decomposing at an anthropological facility has been identified as an important indicator for postmortem interval estimates using machine-learning algorithms13. However, our results do not align with a previous study that documented an increase in abundance of Firmicutes (Clostridium and Lactobacillus) from mouth samples. The lack of continuity from the results could stem from the small sample size (two bodies) and the sample collection times, as microbial communities were compared at pre-bloat to end of bloat during decomposition12. Nor were our results congruent with another study documenting an increase in Actinobacteria and Firmicutes from the skin microbial communities in later decomposition (four bodies decomposing up to 82 days at an anthropological research facility)10. Finally, H. parainfluenzae was identified to be a potential bioindicator using ANCOM tests, specifically in the mouth less than 48 h after death. While no study to date has indicated this taxon as a potential bioindicator of postmortem microbial communities, the genus Haemophilius is part of the normal bacterial flora of the upper respiratory tract45, but can be considered opportunistic pathogens46,47,48, and H. parainfluenzae has been found in the oral cavities of patients living without periodontal disease49. Thus, it is not surprising this taxon commonly found in the mouth of living individuals was an indicator of postmortem intervals less than two days after death.

Constituents of the living host microbial communities, such as Clostridium spp., Streptococci, and the Enterobacteria, have been found to be viable up to 48–72 hours after host death50. Further, we observed members of Enterobacteriaceae that were OTUs most frequently correlated to cell motility. This result is not surprising given the increased relative abundance of Enterobacteriaceae and other λ-Proteobacteria as decomposition progressed. Predominant taxa within Enterobacteriaceae have swarming behavior, including Salmonella and Proteus51. Thus, it is possible that swarming members of Enterobacteriaceae may be outcompeting other microbial communities at later postmortem intervals. Additionally, the predicted functional profiles suggest the taxa detected two days after death (chemotaxis, motility proteins, and flagellar assembly) may have competitive advantages, such as niche colonization or nutrient acquisition, that result in reduced diversity, yet increased motility as determined by in silico functional pathways. Hence, it is not unreasonable to postulate a better understanding of the postmortem microbial communities could provide invaluable insight to human health for the living; assuming that there is not significant change within 24–48 h after death, such as extreme temperature changes that could impact the growth of particular microbial taxa52.

Antemortem and Postmortem Microbiome Linkages

Researchers seeking to better understand the postmortem microbial community dynamics must first determine whether the postmortem microbiota adequately represent microbial populations colonizing the human body prior to death. If this statement holds true, this under-utilized resource could act as a widespread tool for assessing antemortem health, as we have argued in this paper. Previous precedent for use of the postmortem microbiome as a surveillance tool for antemortem health conditions has occurred in tuberculosis research and viral infections53,54,55. We found evidence that cases with heart disease had decreased microbial community diversity (Fig. 3A). This suggests that individuals with heart disease have a reduced microbial configuration, and thus this chronic health condition may be impacting the host microbial biodiversity. As chronic health conditions have been previously reported to impact microbial diversity in living individuals2,56,57. The results of a separate binomial logistic regression for violent deaths (e.g., blunt force trauma, gunshot wound, vehicular accident) suggest that cases resulting from violent deaths had increased phylogenetic diversity (Fig. 3C). We recognize that heart disease typically appears later in life (median age = 53 years) and is chronic, while cases from violent deaths tended to be younger individuals (median age = 38 years) (Table S6), and thus may be a correlating factor contributing to the trends observed. Additional studies that follow microbial communities near, at and after death are necessary to more quantitatively link the associations we report here; however, the logistics of such studies will be very challenging.

Based on these results, we are suggesting that utilization of the microbiome after host death could be an important step toward human health surveillance among other practical applications, mainly forensics, biosecurity and precision medicine. While assessing changes in the postmortem microbiome within the first 24 h of death would be ideal, this would require waiting for death and sampling immediately after death, circumstances of substantial ethical concern and logistic difficulties. However, the alternative approach is to incorporate microbiome sampling during death investigation58, where samples for microbes are often routinely collected to fulfill forensic microbiological protocols, including but not limited to detection and confirmation of microbes of clinical importance (e.g., bacterial meningitis), or biological agents in suspected biocrimes (e.g., Bacillus anthracis). This is the approach that we took for this study, and then identified changes in the microbiome within and after 24 h of death.

To determine the potential of the postmortem microbiome as a health surveillance tool, it is important to first characterize how the microbial communities vary among different postmortem time frames and anatomic areas and identify major changes related to time since death. To assess this potential use, it was important to model the factors and consistency of mechanisms that mediated microbial community dynamics (structure and predictive function) after host death. The detection of potential pathogens and community stability suggests a carryover between living and deceased host microbiota within 24 h of death. Hence, we argue the postmortem microbiota has the promise to identify disease entities that often remain unknown after death, and identifying these biomarker(s) of antemortem health condition would overcome challenges associated with microbiome studies of the living (e.g., limited or targeted sample size and non-invasive sampling). As this and future datasets expand, it is conceivable that resulting data from the postmortem microbiota could provide insights into the health of the community, and even public health intervention if warranted.

Additionally, the data suggest that perhaps individual taxon biomarker(s) will not be as strong of an indicator of health condition when compared to the community of microorganisms associated with the individual (e.g., diversity). However, we detected bacteria in the first 24 h after death that are commonly found in the human microbiota of living individuals1,59. This carryover in taxa is important because it indicates a link between ante- and postmortem (<24 h PMI) microbial communities. For example, there were 83 cases with Streptococccus: 59 of them had Streptococccus in individuals without heart disease, while 24 had Streptococccus in cases with heart disease. Haemophilus and Fusobacterium were two-fold more abundant in healthy individuals where as Rothia was 0.09 times more abundant in heart disease cases. Many species of Rothia are opportunistic pathogens23,60, so it is possible we are detecting infections of individuals that correspond to the subtle abundance increases in unhealthy individuals. Ultimately, we anticipate that the postmortem microbiota could become a valuable resource for informing human health outcomes.

Potential Societal Impacts from the Postmortem Microbiome

Information from our current dataset could have a direct effect on living individuals by providing a means to broadly survey dysbiotic microbiomes associated with human health status, including chronic conditions, such as obesity, diabetes, and asthma, and helping develop strategies for delivering medical care to diverse or low socioeconomic status populations1,59. Here we show that the microbial communities change with time since estimated death, and the consortia within 24 h of death likely represent antemortem health conditions and dysbiosis. This community stability within the first 24–48 h after host death could be of importance for human health and pathogen surveillance and forensics, especially with bacterial species that are difficult to culture with conventional methods. After 48 h the value of postmortem communities for health surveillance may become more limited in this context. Our results indicate the postmortem microbiome could be used as a tool to conduct surveys beneficial to public health, especially in sociocultural areas where there has been consistent failure to understand health states of medically underserved population.

Overall, our study shows that postmortem microbial community dynamics from an urban population reflects a range of human demographics, sociocultural conditions, and life styles. Previous work has provided longitudinal characterizations of the host postmortem microbiota in controlled environments (e.g., anthropological research facility), where body donations are a priori selected based on stringent criteria. These studies often encompass a limited population (up to 50 cases), thus due to the size of our large-scale dataset it allows a robust characterization of the microbial communities after death, and provide data from wide swath of the population not often studied in human microbiome work. Therefore, our dataset contributes empirical evidence that advances the understanding of the postmortem microbiota and their function in a variable population. Further, we postulate the postmortem microbiome, within 24–48 h of death, is a reflection of the host microbiome preceding death. We identified a postmortem microbiota metric as a significant predictor of an important antemortem health status (and the leading cause of death in the US) – heart disease. Thus, this novel approach may provide a comprehensive tool with utility to indicate the state of human health in a way that can be sampled during clinical investigation in a range of deaths, from chronic and natural to sudden and violent.