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Microbial communities associated with the human body are highly dynamic and reflect the host environment and lifestyle over time. Studies show death is no exception, with data demonstrating similar antemortem and postmortem microbiomes up to 48 h following death. These predictable microbial biomarkers can inform death investigation by helping to estimate the postmortem interval and build models to identify cause and manner of death. However, no attempts have been made to model potential microbial biomarkers in pediatric (≤2 years) deaths. This study provided a cross-sectional survey of the microbiota of 53 pediatric cases (black, white, both sexes) seen in Wayne County, Michigan. Autopsy cases represented accidents, homicides, or natural causes. Postmortem microbiome were collected by swabbing the eyes, ears, nose, mouth, umbilicus, brain, rectum, trabecular space, and cardiac blood. 16S rRNA sequence analyses indicated that sex, race, age, body site, and manner of death (MOD) had significant effects on microbiome composition, with significant interactions among MOD, race, and age. Amplicon sequence variants identified intra- and interhost dispersion of the postmortem microbiome depending on death circumstance. Among manners of death, non-accidental deaths were significantly distinct from all other deaths, and among body sites the rectum was distinct in its microbial composition. There is a real need for robust postmortem microbiome before it can be standardized as a practical tool for use in forensic investigation or public health. These results inform postmortem microbial variability during pediatric death investigation that contributes to a larger effort to understand the postmortem microbiome., (© 2024 The Author(s). Journal of Forensic Sciences published by Wiley Periodicals LLC on behalf of American Academy of Forensic Sciences.)

Predicting the outbreak of infectious diseases and designing appropriate preventive health actions require interdisciplinary research into the processes that drive exposure to and transmission of disease agents. In the case of mycobacterial diseases, the epidemiological understanding of the scientific community hitherto was based on the clinical studies of infections in vertebrates. To evaluate the information gained by comprehensively accounting for the ecological and evolutionary constraints, we conducted literature searches assessing the role of mycobacteria interactions with non-vertebrate species in the origin of their pathogenicity and variations in disease risk. The reviewed literature challenges the current theory of person-to-person transmission for several mycobacterial infections. Furthermore, the findings suggest that diverse non-vertebrate organisms influence virulence, mediate transmission, and contribute to pathogen abundance in relation to vertebrate exposure. We advocate that an ecological and evolutionary framework provides novel insights to support a more comprehensive understanding of the prevention and management of diseases in vertebrates., Competing Interests: Declaration of interests We declare no competing interests. The content of the article is the sole responsibility of the authors, with no role played by the funding agencies., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Pathogens such as bacteria, fungi and viruses are important components of soil and aquatic communities, where they can benefit from decaying and living organic matter, and may opportunistically infect human and animal hosts. One-third of human infectious diseases is constituted by sapronotic disease agents that are natural inhabitants of soil or aquatic ecosystems. They are capable of existing and reproducing in the environment outside of the host for extended periods of time. However, as ecological research on sapronosis is infrequent and epidemiological models are even rarer, very little information is currently available. Their importance is overlooked in medical and veterinary research, as well as the relationships between free environmental forms and those that are pathogenic. Here, using dynamical models in realistic aquatic metacommunity systems, we analyze sapronosis transmission, using the human pathogen Mycobacterium ulcerans that is responsible for Buruli ulcer. We show that the persistence of bacilli in aquatic ecosystems is driven by a seasonal upstream supply, and that the attachment and development of cells to aquatic living forms is essential for such pathogen persistence and population dynamics. Our work constitutes the first set of metacommunity models of sapronotic disease transmission, and is highly flexible for adaptation to other types of sapronosis. The importance of sapronotic agents on animal and human disease burden needs better understanding and new models of sapronosis disease ecology to guide the management and prevention of this important group of pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

After death, microbes (including bacteria and fungi) colonize carrion from a variety of sources during the decomposition process. The predictable succession of microbes could be useful for forensics, such as postmortem submersion interval estimation (PMSI) for aquatic deaths. However, gaps exist in our understanding of microbial succession on submerged bone, particularly regarding longer-term decomposition (>1 year), fungal composition, and differences between internal and external microbial communities. To further explore this potential forensic tool, we described the postmortem microbial communities (bacteria and fungi) on and within submerged bones using targeted amplicon sequencing. We hypothesized predictable successional patterns of microbial colonization would be detected on the surface and within submerged bones, which would eventually converge to a similar microbial community. To best replicate forensic contexts, we sampled bones from replicate swine (Sus scrofa domesticus) carcasses submerged in a freshwater pond, every three months for nearly two years. Microbial bone (internal vs. external) community structure (taxa abundance and diversity) of bones differed for both bacteria and fungi, but internal and external communities did not converge to a similar structure. PMSI estimation models built with random forest regression of postmortem microbiomes were highly accurate (>80% variation explained in PMSI) and showed promise for forensic purposes. Overall, we provide further evidence that internal and external bone microbial communities submerged in an aquatic habitat are distinct and each community undergoes predictable succession, demonstrating potential utility in forensics for modeling PMSI in unattended deaths and/or cold cases., (© 2022 American Academy of Forensic Sciences.)

Understanding the interactions of ecosystems, humans and pathogens is important for disease risk estimation. This is particularly true for neglected and newly emerging diseases where modes and efficiencies of transmission leading to epidemics are not well understood. Using a model for other emerging diseases, the neglected tropical skin disease Buruli ulcer (BU), we systematically review the literature on transmission of the etiologic agent, Mycobacterium ulcerans (MU), within a One Health/EcoHealth framework and against Hill's nine criteria and Koch's postulates for making strong inference in disease systems. Using this strong inference approach, we advocate a null hypothesis for MU transmission and other understudied disease systems. The null should be tested against alternative vector or host roles in pathogen transmission to better inform disease management. We propose a re-evaluation of what is necessary to identify and confirm hosts, reservoirs and vectors associated with environmental pathogen replication, dispersal and transmission; critically review alternative environmental sources of MU that may be important for transmission, including invertebrate and vertebrate species, plants and biofilms on aquatic substrates; and conclude with placing BU within the context of other neglected and emerging infectious diseases with intricate ecological relationships that lead to disease in humans, wildlife and domestic animals., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

The postmortem microbiome plays an important functional role in host decomposition after death. Postmortem microbiome community successional patterns are specific to body site, with a significant shift in composition 48 h after death. While the postmortem microbiome has important forensic applications for postmortem interval estimation, it also has the potential to aid in manner of death (MOD) and cause of death (COD) determination as a reflection of antemortem health status. To further explore this association, we tested beta-dispersion, or the variability of microbiomes within the context of the "Anna Karenina Principle" (AKP). The foundational principle of AKP is that stressors affect microbiomes in unpredictable ways, which increases community beta-dispersion. We hypothesized that cases with identified M/CODs would have differential community beta-dispersion that reflected antemortem conditions, specifically that cardiovascular disease and/or natural deaths would have higher beta-dispersion compared to other deaths (e.g., accidents, drug-related deaths). Using a published microbiome data set of 188 postmortem cases (five body sites per case) collected during routine autopsy in Wayne County (Detroit), MI, we modeled beta-dispersion to test for M/COD associations a priori . Logistic regression models of beta-dispersion and case demographic data were used to classify M/COD. We demonstrated that beta-dispersion, along with case demographic data, could distinguish among M/COD - especially cardiovascular disease and drug related deaths, which were correctly classified in 79% of cases. Binary logistic regression models had higher correct classifications than multinomial logistic regression models, but changing the defined microbial community (e.g., full vs. non-core communities) used to calculate beta-dispersion overall did not improve model classification or M/COD. Furthermore, we tested our analytic approach on a case study that predicted suicides from other deaths, as well as distinguishing MOD (e.g., homicides vs. suicides) within COD (e.g., gunshot wound). We propose an analytical workflow that combines postmortem microbiome indicator taxa, beta-dispersion, and case demographic data for predicting MOD and COD classifications. Overall, we provide further evidence the postmortem microbiome is linked to the host's antemortem health condition(s), while also demonstrating the potential utility of including beta-dispersion (a non-taxon dependent approach) coupled with case demographic data for death determination., (Copyright © 2020 Kaszubinski, Pechal, Smiles, Schmidt, Jordan, Meek and Benbow.)

Louse flies (Diptera: Hippoboscidae) are obligate ectoparasites that often cause behavioral, pathogenic, and evolutionary effects on their hosts. Interactions between ectoparasites and avian hosts, especially migrating taxa, may influence avian pathogen spread in tropical and temperate ecosystems and affect long-term survival, fitness and reproductive success. The purpose of this study was to characterize the vector-associated microbiome of ectoparasitic louse flies feeding on migrating raptors over the fall migration period. Surveys for louse flies occurred during fall migration (2015-2016) at a banding station in Pennsylvania, United States; flies were collected from seven species of migrating raptors, and we sequenced their microbial (bacteria and archaea) composition using high-throughput targeted amplicon sequencing of the 16S rRNA gene (V4 region). All louse flies collected belonged to the same species, Icosta americana. Our analysis revealed no difference in bacterial communities of louse flies retrieved from different avian host species. The louse fly microbiome was dominated by a primary endosymbiont, suggesting that louse flies maintain a core microbial structure despite receiving blood meals from different host species. Thus, our findings highlight the importance of characterizing both beneficial and potentially pathogenic endosymbionts when interpreting how vector-associated microbiomes may impact insect vectors and their avian hosts., Competing Interests: The authors have declared that no competing interests exist.

Necrophagous insects play an important role in the decomposition of vertebrate carrion. The documented colonization, development, and succession of blow flies (Diptera: Calliphoridae) and other arthropods on decomposing carcasses make their communities relevant for use in decomposition ecology and forensic investigations. This relevance relies on the local pool of species available to colonize a carcass, but such community level survey data are not always available. The objective of this research was to conduct a baseline survey of adult Calliphoridae communities from urban-rural land use types in the Great Lakes region. To test how adult blow fly distribution varies with changing landscape in Mid-Michigan, sampling with baited jars and hanging traps was implemented over the summer months of June, July, and August 2017. To determine how blow fly communities differed in urban to rural land cover, seven cities were selected with site locations ranging from high intensity developed areas to cultivated crop fields. Over 97,000 individual flies were captured represented by 11 Calliphoridae species. The adult Calliphoridae communities were primarily structured by land use type and month of collection, with these two factors interacting, suggesting that the effect of location varied by time of year. The two most abundant species, Phormia regina (Meigen) and Lucilia sericata (Meigen), cumulatively comprised 88.5% adult flies from Mid-Michigan. These findings provide a baseline database of Great Lakes Calliphoridae, with potential use in forensic research and casework., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories. These differences can potentially affect microbial community composition and downstream analyses. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, varying minimum library size or minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that three open-source bioinformatic pipelines, MG-RAST, mothur, and QIIME2, had significant differences in relative abundance, alpha-diversity, and beta-diversity, despite the same input data. Increasing minimum library size and sample size increased the number of low-abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice affect results in important ways. Given the growing potential application of forensic microbiology to the criminal justice system, continued research on standardizing computational methodology will be important for downstream applications., (© 2019 American Academy of Forensic Sciences.)

Multiple methods have been proposed to provide accurate time since death estimations, and recently, the discovery of bacterial community turnover during decomposition has shown itself to have predictable patterns that may prove useful. In this study, we demonstrate the use of metatranscriptomics from the postmortem microbiome to simultaneously obtain community structure and functional data across postmortem intervals (PMIs). We found that bacterial succession patterns reveal similar trends as detected through DNA analysis, such as increasing Clostridiaceae as decomposition occurs, strengthening the reliability of total RNA community analyses. We also provide one of the first analyses of RNA transcripts to characterize bacterial metabolic pathways during decomposition. We found distinct pathways, such as amino acid metabolism, to be strongly up-regulated with increasing PMIs. Elucidating the metabolic activity of postmortem microbial communities provides the first steps to discovering postmortem functional biomarkers since functional redundancy across bacteria may reduce host individual microbiome variability., (© 2019 American Academy of Forensic Sciences.)

The decomposition of animal biomass (carrion) contributes to the recycling of energy and nutrients through ecosystems. Whereas the role of plant decomposition in ecosystems is broadly recognised, the significance of carrion to ecosystem functioning remains poorly understood. Quantitative data on carrion biomass are lacking and there is no clear pathway towards improved knowledge in this area. Here, we present a framework to show how quantities derived from individual carcasses can be scaled up using population metrics, allowing for comparisons among ecosystems and other forms of biomass. Our framework facilitates the generation of new data that is critical to building a quantitative understanding of the contribution of carrion to trophic processes and ecosystem stocks and flows., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

Background: The postmortem microbiome can provide valuable information to a death investigation and to the human health of the once living. Microbiome sequencing produces, in general, large multi-dimensional datasets that can be difficult to analyze and interpret. Machine learning methods can be useful in overcoming this analytical challenge. However, different methods employ distinct strategies to handle complex datasets. It is unclear whether one method is more appropriate than others for modeling postmortem microbiomes and their ability to predict attributes of interest in death investigations, which require understanding of how the microbial communities change after death and may represent those of the once living host., Methods and Findings: Postmortem microbiomes were collected by swabbing five anatomical areas during routine death investigation, sequenced and analyzed from 188 death cases. Three machine learning methods (boosted algorithms, random forests, and neural networks) were compared with respect to their abilities to predict case attributes: postmortem interval (PMI), location of death, and manner of death. Accuracy depended on the method used, the numbers of anatomical areas analyzed, and the predicted attribute of death., Conclusions: All algorithms performed well but with distinct features to their performance. Xgboost often produced the most accurate predictions but may also be more prone to overfitting. Random forest was the most stable across predictions that included more anatomic areas. Analysis of postmortem microbiota from more than three anatomic areas appears to yield limited returns on accuracy, with the eyes and rectum providing the most useful information correlating with circumstances of death in most cases for this dataset., Competing Interests: The authors have declared that no competing interests exist.

Population-based public health data on antibiotic resistance gene carriage is poorly surveyed. Research of the human microbiome as an antibiotic resistance reservoir has primarily focused on gut associated microbial communities, but data have shown more widespread microbial colonization across organs than originally believed, with organs previously considered as sterile being colonized. Our study demonstrates the utility of postmortem microbiome sampling during routine autopsy as a method to survey antibiotic resistance carriage in a general population. Postmortem microbial sampling detected pathogens of public health concern including genes for multidrug efflux pumps, carbapenem, methicillin, vancomycin, and polymixin resistances. Results suggest that postmortem assessments of host-associated microbial communities are useful in acquiring community specific data while reducing selective-participant biases., Competing Interests: The authors have declared that no competing interests exist.

Ephemeral aquatic habitats and their associated microbial communities (microbiomes) play important roles in the growth and development of numerous aquatic insects, including mosquitoes (Diptera). Biological control agents, such as Bacillus thuringiensis israelensis (Bti) or insect growth regulators (e.g., methoprene), are commonly used to control mosquitoes in these habitats. However, it is unknown how commonly used control compounds affect the mosquito internal microbiome and potentially alter their life history traits. The objectives of this study were threefold: characterize the internal microbiota of Aedes larvae (Culicidae) in ephemeral forested mosquito habitat using high-throughput amplicon based sequencing, assess how mosquito control treatments affect the internal microbial communities of larval mosquitoes, and determine if changes to the microbiome resulted from direct or indirect treatment effects. The larval microbiome varied in community composition and diversity with development stage and treatment, suggesting potential effects of control compounds on insect microbial ecology. While microbial community differences due to Bti treatment were a result of indirect effects on larval development, methoprene had significant impacts on bacterial and algal taxa that could not be explained by indirect treatment effects. These results provide new information on the interactions between pesticide treatments and insect microbial communities.

The microbiome plays many roles in human health, often through the exclusive lens of clinical interest. The inevitable end point for all living hosts, death, has its own altered microbiome configurations. However, little is understood about the ecology and changes of microbial communities after death, or their potential utility for understanding the health condition of the recently living. Here we reveal distinct postmortem microbiomes of human hosts from a large-scale survey of death cases representing a predominantly urban population, and demonstrated these microbiomes reflected antemortem health conditions within 24-48 hours of death. Our results characterized microbial community structure and predicted function from 188 cases representing a cross-section of an industrial-urban population. We found strong niche differentiation of anatomic habitat and microbial community turnover based on topographical distribution. Microbial community stability was documented up to two days after death. Additionally, we observed a positive relationship between cell motility and time since host death. Interestingly, we discovered evidence that microbial biodiversity is a predictor of antemortem host health condition (e.g., heart disease). These findings improve the understanding of postmortem host microbiota dynamics, and provide a robust dataset to test the postmortem microbiome as a tool for assessing health conditions in living populations.

As vertebrate carrion decomposes, there is a release of nutrient-rich fluids into the underlying soil, which can impact associated biological community structure and function. How these changes alter soil biogeochemical cycles is relatively unknown and may prove useful in the identification of carrion decomposition islands that have long lasting, focal ecological effects. This study investigated the spatial (0, 1, and 5 m) and temporal (3-732 days) dynamics of human cadaver decomposition on soil bacterial and arthropod community structure and microbial function. We observed strong evidence of a predictable response to cadaver decomposition that varies over space for soil bacterial and arthropod community structure, carbon (C) mineralization and microbial substrate utilization patterns. In the presence of a cadaver (i.e., 0 m samples), the relative abundance of Bacteroidetes and Firmicutes was greater, while the relative abundance of Acidobacteria, Chloroflexi, Gemmatimonadetes, and Verrucomicrobia was lower when compared to samples at 1 and 5 m. Micro-arthropods were more abundant (15 to 17-fold) in soils collected at 0 m compared to either 1 or 5 m, but overall, micro-arthropod community composition was unrelated to either bacterial community composition or function. Bacterial community structure and microbial function also exhibited temporal relationships, whereas arthropod community structure did not. Cumulative precipitation was more effective in predicting temporal variations in bacterial abundance and microbial activity than accumulated degree days. In the presence of the cadaver (i.e., 0 m samples), the relative abundance of Actinobacteria increased significantly with cumulative precipitation. Furthermore, soil bacterial communities and C mineralization were sensitive to the introduction of human cadavers as they diverged from baseline levels and did not recover completely in approximately 2 years. These data are valuable for understanding ecosystem function surrounding carrion decomposition islands and can be applicable to environmental bio-monitoring and forensic sciences.

Previous postmortem microbiome studies have focused on characterizing taxa turnover during an undisturbed decomposition process. How coexisting conditions (e.g., frozen, buried, burned) affect the human microbiome at the time of discovery is less well understood. Microbiome data were collected from two pediatric cases at the Wayne County Medical Examiner in Michigan. The bodies were found frozen, hidden in a freezer for an extended time. Microbial communities were sampled from six external anatomic locations at three time points during the thawing process, prior to autopsy. The 16S rRNA V4 gene amplicon region was sequenced using high-throughput sequencing (Illumina MiSeq). Microbial diversity increased, and there was a distinct shift in microbial community structure and abundance throughout the thawing process. Overall, these data demonstrate that the postmortem human microbiome changes during the thawing process, and have important forensic implications when bodies have been substantially altered, modified, and concealed after death., (© 2017 American Academy of Forensic Sciences.)

Insects associated with carrion are critical to the decomposition process and nutrient cycling in ecosystems. Yet the communities of insects associated with carrion vary between locations, and detailed case studies are necessary for identifying differences and similarities among contrasting habitats. In this study, we examined temporal changes in the crawling insect community collected from rabbit carcasses placed in contrasting grassland and tree habitats in southeastern Australia. We collected 18,400 adult insects, including 22 species of fly, 57 species of beetle, and 37 species of ant. We found significant effects of habitat type and time, but not their interaction, on the composition of the entire insect community. Several ant species showed early and rapid colonization and highest abundances during early stages of decay, including Iridomyrmex purpureus (Smith, 1858) under trees, and Iridomyrmex rufoniger (Lowne, 1865) and Rhytidoponera metallica (Smith, 1858) in grassland. We found that most fly species showed highest abundance during active decay, but Chrysomya varipes (Macquart 1851) was more abundant under trees than in grassland during this time. Beetles peaked during active or advanced decay stages, with Saprinus and Omorgus the most abundant genera. Our study demonstrates that strong replication of contrasting environmental treatments can reveal new information on habitat preferences of important carrion insect species. The numerical dominance of ants early in decomposition has implications for insect community structure via potential competitive interactions with flies, and should be more rigorously examined in future carrion studies., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Cuticular hydrocarbons (CHC) have been successfully used in the field of forensic entomology for identifying and ageing forensically important blowfly species, primarily in the larval stages. However in older scenes where all other entomological evidence is no longer present, Calliphoridae puparial cases can often be all that remains and therefore being able to establish the age could give an indication of the PMI. This paper examined the CHCs present in the lipid wax layer of insects, to determine the age of the cases over a period of nine months. The two forensically important species examined were Calliphora vicina and Lucilia sericata. The hydrocarbons were chemically extracted and analysed using Gas Chromatography - Mass Spectrometry. Statistical analysis was then applied in the form of non-metric multidimensional scaling analysis (NMDS), permutational multivariate analysis of variance (PERMANOVA) and random forest models. This study was successful in determining age differences within the empty cases, which to date, has not been establish by any other technique.

Common forensic entomology practice has been to collect the largest Diptera larvae from a scene and use published developmental data, with temperature data from the nearest weather station, to estimate larval development time and post-colonization intervals (PCIs). To evaluate the accuracy of PCI estimates among Calliphoridae species and spatially distinct temperature sources, larval communities and ambient air temperature were collected at replicate swine carcasses (N = 6) throughout decomposition. Expected accumulated degree hours (ADH) associated with Cochliomyia macellaria and Phormia regina third instars (presence and length) were calculated using published developmental data sets. Actual ADH ranges were calculated using temperatures recorded from multiple sources at varying distances (0.90 m-7.61 km) from the study carcasses: individual temperature loggers at each carcass, a local weather station, and a regional weather station. Third instars greatly varied in length and abundance. The expected ADH range for each species successfully encompassed the average actual ADH for each temperature source, but overall under-represented the range. For both calliphorid species, weather station data were associated with more accurate PCI estimates than temperature loggers associated with each carcass. These results provide an important step towards improving entomological evidence collection and analysis techniques, and developing forensic error rates.

Microbially mediated mechanisms of human decomposition begin immediately after death, and are a driving force for the conversion of a once living organism to a resource of energy and nutrients. Little is known about post-mortem microbiology in cadavers, particularly the community structure of microflora residing within the cadaver and the dynamics of these communities during decomposition. Recent work suggests these bacterial communities undergo taxa turnover and shifts in community composition throughout the post-mortem interval. In this paper we describe how the microbiome of a living host changes and transmigrates within the body after death thus linking the microbiome of a living individual to post-mortem microbiome changes. These differences in the human post-mortem from the ante-mortem microbiome have demonstrated promise as evidence in death investigations. We investigated the post-mortem structure and function dynamics of Staphylococcus aureus and Clostridium perfringens after intranasal inoculation in the animal model Mus musculus L. (mouse) to identify how transmigration of bacterial species can potentially aid in post-mortem interval estimations. S. aureus was tracked using in vivo and in vitro imaging to determine colonization routes associated with different physiological events of host decomposition, while C. perfringens was tracked using culture-based techniques. Samples were collected at discrete time intervals associated with various physiological events and host decomposition beginning at 1h and ending at 60 days post-mortem. Results suggest that S. aureus reaches its highest concentration at 5-7 days post-mortem then begins to rapidly decrease and is undetectable by culture on day 30. The ability to track these organisms as they move in to once considered sterile space may be useful for sampling during autopsy to aid in determining post-mortem interval range estimations, cause of death, and origins associated with the geographic location of human remains during death investigations., (Published by Elsevier Ireland Ltd.)

Carrion decomposition is driven by complex relationships that affect necrobiome community (i.e. all organisms and their genes associated with a dead animal) interactions, such as insect species arrival time to carrion and microbial succession. Little is understood about how microbial communities interact with invertebrates at the aquatic-terrestrial habitat interface. The first objective of the study was to characterize internal microbial communities using high-throughput sequencing of 16S rRNA gene amplicons for aquatic insects (three mayfly species) in streams with salmon carcasses compared with those in streams without salmon carcasses. The second objective was to assess the epinecrotic microbial communities of decomposing salmon carcasses (Oncorhynchus keta) compared with those of terrestrial necrophagous insects (Calliphora terraenovae larvae and adults) associated with the carcasses. There was a significant difference in the internal microbiomes of mayflies collected in salmon carcass-bearing streams and in non-carcass streams, while the developmental stage of blow flies was the governing factor in structuring necrophagous insect internal microbiota. Furthermore, the necrophagous internal microbiome was influenced by the resource on which the larvae developed, and changes in the adult microbiome varied temporally. Overall, these carrion subsidy-driven networks respond to resource pulses with bottom-up effects on consumer microbial structure, as revealed by shifting communities over space and time., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Genomic studies have estimated there are approximately 10(3)-10(6) bacterial species per gram of soil. The microbial species found in soil associated with decomposing human remains (gravesoil) have been investigated and recognized as potential molecular determinants for estimates of time since death. The nascent era of high-throughput amplicon sequencing of the conserved 16S ribosomal RNA (rRNA) gene region of gravesoil microbes is allowing research to expand beyond more subjective empirical methods used in forensic microbiology. The goal of the present study was to evaluate microbial communities and identify taxonomic signatures associated with the gravesoil human cadavers. Using 16S rRNA gene amplicon-based sequencing, soil microbial communities were surveyed from 18 cadavers placed on the surface or buried that were allowed to decompose over a range of decomposition time periods (3-303 days). Surface soil microbial communities showed a decreasing trend in taxon richness, diversity, and evenness over decomposition, while buried cadaver-soil microbial communities demonstrated increasing taxon richness, consistent diversity, and decreasing evenness. The results show that ubiquitous Proteobacteria was confirmed as the most abundant phylum in all gravesoil samples. Surface cadaver-soil communities demonstrated a decrease in Acidobacteria and an increase in Firmicutes relative abundance over decomposition, while buried soil communities were consistent in their community composition throughout decomposition. Better understanding of microbial community structure and its shifts over time may be important for advancing general knowledge of decomposition soil ecology and its potential use during forensic investigations.

Biofilms are a ubiquitous formation of microbial communities found on surfaces in aqueous environments. These structures have been investigated as biomonitoring indicators for stream heath, and here were used for the potential use in forensic sciences. Biofilm successional development has been proposed as a method to determine the postmortem submersion interval (PMSI) of remains because there are no standard methods for estimating the PMSI and biofilms are ubiquitous in aquatic habitats. We sought to compare the development of epinecrotic (biofilms on Sus scrofa domesticus carcasses) and epilithic (biofilms on unglazed ceramic tiles) communities in two small streams using bacterial automated ribosomal intergenic spacer analysis. Epinecrotic communities were significantly different from epilithic communities even though environmental factors associated with each stream location also had a significant influence on biofilm structure. All communities at both locations exhibited significant succession suggesting that changing communities throughout time is a general characteristic of stream biofilm communities. The implications resulting from this work are that epinecrotic communities have distinctive shifts at the first and second weeks, and therefore the potential to be used in forensic applications by associating successional changes with submersion time to estimate a PMSI. The influence of environmental factors, however, indicates the lack of a successional pattern with the same organisms and a focus on functional diversity may be more applicable in a forensic context., Competing Interests: The authors declare no conflict of interest.

Human remains can be discovered in freshwater or marine ecosystems, circumstances where insects and other invertebrates have infrequently been used for understanding the time of postmortem submersion. In this study, the identification and succession of epinecrotic bacterial communities on vertebrate remains were described during decomposition in a temperate headwater stream during two seasons (summer and winter). Bacterial communities were characterized with 454 pyrosequencing and analyzed at phyletic and generic taxonomic resolutions. There was a significant increase in genera richness over decomposition during both seasons. Additionally, multivariate statistical modeling revealed significant differences in bacterial communities between seasons at both taxonomic resolutions and siginificant genera differences among sampling days within each season, suggesting a succession of these communities. These data are the first to describe aquatic bacterial succession using high-throughput metagenomic sequencing on vertebrate remains submerged in a freshwater habitat, and provide initial evidence for their potential use in forensic investigations., (© 2015 American Academy of Forensic Sciences.)

During a forensic investigation, the presence of physical marks on human remains can influence the interpretation of events related to the death of an individual. Some tissue injury on human remains can be misinterpreted as ante- or peri-mortem wounds by an investigator when in reality the markings resulted from post-mortem arthropod activity. Unusual entomological data were collected during a study examining the decomposition of a set of human remains in San Marcos, Texas. An adult female Pediodectes haldemani (Girard) (Orthoptera: Tettigoniidae) and an Armadillidium cf. vulgare (Isopoda: Armadilidiidae) were documented feeding on the remains. Both arthropods produced physical marks or artifacts on the remains that could be misinterpreted as attack, abuse, neglect, or torture. Additionally, red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae), were observed constructing structures in the mark produced by the P. haldemani feeding. These observations provide insight into the potential of post-mortem arthropod damage to human remains, which previously had not been described for these taxa, and therefore, physical artifacts on any remains found in similar circumstances may result from arthropod activity and not ante- or peri-mortem wounds., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Blow flies (Diptera: Calliphoridae) are typically the first insects to arrive at human remains and carrion. Predictable succession patterns and known larval development of necrophagous insects on vertebrate remains can assist a forensic entomologist with estimates of a minimum post-mortem interval (PMImin) range. However, adult blow flies are infrequently used to estimate the PMImin, but rather are used for a confirmation of larval species identification. Cuticular hydrocarbons have demonstrated potential for estimating adult blow fly age, as hydrocarbons are present throughout blow fly development, from egg to adult, and are stable structures. The goal of this study was to identify hydrocarbon profiles associated with the adults of a North American native blow fly species, Cochliomyia macellaria (Fabricius) and a North American invasive species, Chrysomya rufifacies (Macquart). Flies were reared at a constant temperature (25°C), a photoperiod of 14:10 (L:D) (h), and were provided water, sugar and powdered milk ad libitum. Ten adult females from each species were collected at day 1, 5, 10, 20, and 30 post-emergence. Hydrocarbon compounds were extracted and then identified using gas chromatography-mass spectrometry (GC-MS) analysis. A total of 37 and 35 compounds were detected from C. macellaria and Ch. rufifacies, respectively. There were 24 and 23 n-alkene and methyl-branched alkane hydrocarbons from C. macellaria and Ch. rufifacies, respectively (10 compounds were shared between species), used for statistical analysis. Non-metric multidimensional scaling analysis and permutational multivariate analysis of variance were used to analyze the hydrocarbon profiles with significant differences (P<0.001) detected among post-emergence age cohorts for each species, and unique hydrocarbon profiles detected as each adult blow fly species aged. This work provides empirical data that serve as a foundation for future research into improving PMImin estimates made by forensic practitioners and potentially increase the use of adult insects during death investigations., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Decomposition studies of vertebrate remains primarily focus on data that can be seen with the naked eye, such as arthropod or vertebrate scavenger activity, with little regard for what might be occurring with the microorganism community. Here, we discuss the necrobiome, or community of organisms associated with the decomposition of remains, specifically, the "epinecrotic" bacterial community succession throughout decomposition of vertebrate carrion. Pyrosequencing was used to (1) detect and identify bacterial community abundance patterns that described discrete time points of the decomposition process and (2) identify bacterial taxa important for estimating physiological time, a time-temperature metric that is often commensurate with minimum post-mortem interval estimates, via thermal summation models. There were significant bacterial community structure differences in taxon richness and relative abundance patterns through the decomposition process at both phylum and family taxonomic classification levels. We found a significant negative linear relationship for overall phylum and family taxon richness as decomposition progressed. Additionally, we developed a statistical model using high throughput sequencing data of epinecrotic bacterial communities on vertebrate remains that explained 94.4 % of the time since placement of remains in the field, which was within 2-3 h of death. These bacteria taxa are potentially useful for estimating the minimum post-mortem interval. Lastly, we provide a new framework and standard operating procedure of how this novel approach of using high throughput metagenomic sequencing has remarkable potential as a new forensic tool. Documenting and identifying differences in bacterial communities is key to advancing knowledge of the carrion necrobiome and its applicability in forensic science.

Microorganisms play a critical role in the decomposition of organic matter, which contributes to energy and nutrient transformation in every ecosystem. Yet, little is known about the functional activity of epinecrotic microbial communities associated with carrion. The objective of this study was to provide a description of the carrion associated microbial community functional activity using differential carbon source use throughout decomposition over seasons, between years and when microbial communities were isolated from eukaryotic colonizers (e.g., necrophagous insects). Additionally, microbial communities were identified at the phyletic level using high throughput sequencing during a single study. We hypothesized that carrion microbial community functional profiles would change over the duration of decomposition, and that this change would depend on season, year and presence of necrophagous insect colonization. Biolog EcoPlates™ were used to measure the variation in epinecrotic microbial community function by the differential use of 29 carbon sources throughout vertebrate carrion decomposition. Pyrosequencing was used to describe the bacterial community composition in one experiment to identify key phyla associated with community functional changes. Overall, microbial functional activity increased throughout decomposition in spring, summer and winter while it decreased in autumn. Additionally, microbial functional activity was higher in 2011 when necrophagous arthropod colonizer effects were tested. There were inconsistent trends in the microbial function of communities isolated from remains colonized by necrophagous insects between 2010 and 2011, suggesting a greater need for a mechanistic understanding of the process. These data indicate that functional analyses can be implemented in carrion studies and will be important in understanding the influence of microbial communities on an essential ecosystem process, carrion decomposition.

Necrophagous invertebrates have been documented to be a predominant driver of vertebrate carrion decomposition; however, very little is understood about the assembly of these communities both within and among seasons. The objective of this study was to evaluate the seasonal differences in insect taxa composition, richness, and diversity on carrion over decomposition with the intention that such data will be useful for refining error estimates in forensic entomology. Sus scrofa (L.) carcasses (n = 3-6, depending on season) were placed in a forested habitat near Xenia, OH, during spring, summer, autumn, and winter. Taxon richness varied substantially among seasons but was generally lower (1-2 taxa) during early decomposition and increased (3-8 taxa) through intermediate stages of decomposition. Autumn and winter showed the highest richness during late decomposition. Overall, taxon richness was higher during active decay for all seasons. While invertebrate community composition was generally consistent among seasons, the relative abundance of five taxa significantly differed across seasons, demonstrating different source communities for colonization depending on the time of year. There were significantly distinct necrophagous insect communities for each stage of decomposition, and between summer and autumn and summer and winter, but the communities were similar between autumn and winter. Calliphoridae represented significant indicator taxa for summer and autumn but replaced by Coleoptera during winter. Here we demonstrated substantial variability in necrophagous communities and assembly on carrion over decomposition and among seasons. Recognizing this variation has important consequences for forensic entomology and future efforts to provide error rates for estimates of the postmortem interval using arthropod succession data as evidence during criminal investigations.

Established procedures for collecting and preserving evidence are essential for all forensic disciplines to be accepted in court and by the forensic community at large. Entomological evidence, such as Diptera larvae, are primarily preserved in ethanol, which can evaporate over time, resulting in the dehydration of specimens. In this study, methods used for rehydrating specimens were compared. The changes in larval specimens with respect to larval length and weight for three forensically important blow fly (Diptera: Calliphoridae) species in North America were quantified. Phormia regina (Meigen), Cochliomyia macellaria (F.), and Chrysomya rufifacies (Macquart) third-instar larvae were collected from various decomposing animals and preserved with three preservation methods (80% ethanol, 70% isopropyl alcohol, and hot-water kill then 80% ethanol). Preservative solutions were allowed to evaporate. Rehydration was attempted with either of the following: 80% ethanol, commercial trisodium phosphate substitute solution, or 0.5% trisodium phosphate solution. All three methods partially restored weight and length of specimens recorded before preservation. Analysis of variance results indicated that effects of preservation, rehydration treatment, and collection animal were different in each species. The interaction between preservative method and rehydration treatment had a significant effect on both P. regina and C. macellaria larval length and weight. In addition, there was a significant interaction effect of collection animal on larval C. macellaria measurements. No significant effect was observed in C. rufifacies larval length or weight among the preservatives or treatments. These methods could be used to establish a standard operating procedure for dealing with dehydrated larval specimens in forensic investigations.