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Distinctive thanatomicrobiome signatures found in the blood and internal organs of humans
... The body thus becomes a huge source of nutrients containing nitrogen, carbon, phosphorus, etc. (Metcalf et al. 2016 a, b). The postmortem microbiome plays an important functional role in the postmortem decomposition of the host (Can et al., 2014).The thanatomicrobiome is therefore composed of certain external commensal microbes that begin to colonize body orifices after death, and of the internal microbial community that colonizes human organs, whereas the necrobiome exclusively defines the external bacterial communities related to a human cadaver (Javan et al., 2016b). Changes in post-mortem microbial communities in a decomposing cadaver generally occur through horizontal migration from the host's digestive tract and through contact with the external environment (Javan et al., 2019;Can et al., 2014;Javan et al., 2016b;Javan et al., 2017). ...
... The postmortem microbiome plays an important functional role in the postmortem decomposition of the host (Can et al., 2014).The thanatomicrobiome is therefore composed of certain external commensal microbes that begin to colonize body orifices after death, and of the internal microbial community that colonizes human organs, whereas the necrobiome exclusively defines the external bacterial communities related to a human cadaver (Javan et al., 2016b). Changes in post-mortem microbial communities in a decomposing cadaver generally occur through horizontal migration from the host's digestive tract and through contact with the external environment (Javan et al., 2019;Can et al., 2014;Javan et al., 2016b;Javan et al., 2017). Bacteria that colonize the surface of the decaying cadaver undergo uncontrolled reproduction, leading to the dormancy of other microbes. ...
... The release of cell contents after lysis significantly alters the composition of human microbial communities. In addition, during this process, certain external microbes begin to colonize the surface and the orifices of the body (Can et al., 2014). After death, failure of the host's immune system allows many germs to proliferate in internal organs, which are considered sterile, within 24 hours (Gevers, 1975;Morris et al., 2006). ...
The circumstances of death and the estimation of the post-mortem interval (PMI) are often a great challenge for scientific and judicial investigators, especially when some time has elapsed since death. Several techniques are used; nevertheless, each presents its own limitations. In the quest for new techniques that are more reliable or at least complementary to those existing and sometimes less expensive, researchers have in recent years turned toward exploring the dynamics of the different microbial communities of a corpse according to their different stages of decomposition. This article summarizes the various works done in the field and shows the different sources of microorganisms in the different parts of the human corpse and their potential interest in the field of forensic medicine.
... Following death, host microbes are involved in the breakdown of tissues and both microbial diversity and composition changes as decomposition progresses (Dent et al. 2004). Microbial communities, mainly bacteria, have been surveyed in different organs and internal body habitats including the eyes (Pechal et al. 2018), nose (Johnson et al. 2016, Pechal et al. 2018, ears (Johnson et al. 2016, Pechal et al. 2018, heart (Can et al. 2014, Javan et al. 2016, Bell et al. 2018, Liu et al. 2020, Lutz et al. 2020, liver (Heimesaat et al. 2012, Can et al. 2014, Javan et al. 2017, Lutz et al. 2020, spleen (Heimesaat et al. 2012, Can et al. 2014, Javan et al. 2016, Lutz et al. 2020), brain (Can et al. 2014, Javan et al. 2016, Liu et al. 2020, Lutz et al. 2020, intestinal tract (Heimesaat et al. 2012, Hauther et al. 2015, Hyde et al. 2015, DeBruyn and Hauther 2017, Pechal et al. 2018, Iancu et al. 2016, Liu et al. 2020, Hu et al. 2021, Li et al. 2021, and mouth (Pechal et al. 2014, Hyde et al. 2015, Javan et al. 2016, Pechal et al. 2018, Dong et al. 2019). These studies have been conducted over time scales ranging from one hour to 30 days (Table 1). ...
... Following death, host microbes are involved in the breakdown of tissues and both microbial diversity and composition changes as decomposition progresses (Dent et al. 2004). Microbial communities, mainly bacteria, have been surveyed in different organs and internal body habitats including the eyes (Pechal et al. 2018), nose (Johnson et al. 2016, Pechal et al. 2018, ears (Johnson et al. 2016, Pechal et al. 2018, heart (Can et al. 2014, Javan et al. 2016, Bell et al. 2018, Liu et al. 2020, Lutz et al. 2020, liver (Heimesaat et al. 2012, Can et al. 2014, Javan et al. 2017, Lutz et al. 2020, spleen (Heimesaat et al. 2012, Can et al. 2014, Javan et al. 2016, Lutz et al. 2020), brain (Can et al. 2014, Javan et al. 2016, Liu et al. 2020, Lutz et al. 2020, intestinal tract (Heimesaat et al. 2012, Hauther et al. 2015, Hyde et al. 2015, DeBruyn and Hauther 2017, Pechal et al. 2018, Iancu et al. 2016, Liu et al. 2020, Hu et al. 2021, Li et al. 2021, and mouth (Pechal et al. 2014, Hyde et al. 2015, Javan et al. 2016, Pechal et al. 2018, Dong et al. 2019). These studies have been conducted over time scales ranging from one hour to 30 days (Table 1). ...
... Following death, host microbes are involved in the breakdown of tissues and both microbial diversity and composition changes as decomposition progresses (Dent et al. 2004). Microbial communities, mainly bacteria, have been surveyed in different organs and internal body habitats including the eyes (Pechal et al. 2018), nose (Johnson et al. 2016, Pechal et al. 2018, ears (Johnson et al. 2016, Pechal et al. 2018, heart (Can et al. 2014, Javan et al. 2016, Bell et al. 2018, Liu et al. 2020, Lutz et al. 2020, liver (Heimesaat et al. 2012, Can et al. 2014, Javan et al. 2017, Lutz et al. 2020, spleen (Heimesaat et al. 2012, Can et al. 2014, Javan et al. 2016, Lutz et al. 2020), brain (Can et al. 2014, Javan et al. 2016, Liu et al. 2020, Lutz et al. 2020, intestinal tract (Heimesaat et al. 2012, Hauther et al. 2015, Hyde et al. 2015, DeBruyn and Hauther 2017, Pechal et al. 2018, Iancu et al. 2016, Liu et al. 2020, Hu et al. 2021, Li et al. 2021, and mouth (Pechal et al. 2014, Hyde et al. 2015, Javan et al. 2016, Pechal et al. 2018, Dong et al. 2019). These studies have been conducted over time scales ranging from one hour to 30 days (Table 1). ...
Vertebrate decomposition results in an ephemeral disturbance of the surrounding environment. Microbial decomposers are recognized as key players in the breakdown of complex organic compounds, controlling carbon and nutrient fate in the ecosystem and potentially serving as indicators of time since death for forensic applications. As a result, there has been increasing attention on documenting the microbial communities associated with vertebrate decomposition, or the 'necrobiome'. These necrobiome studies differ in the vertebrate species, microhabitats (e.g. skin vs. soil), and geographic locations studied, but many are narrowly focused on the forensic application of microbial data, missing the larger opportunity to understand the ecology of these communities. To further our understanding of microbial dynamics during vertebrate decomposition and identify knowledge gaps, there is a need to assess the current works from an ecological systems perspective. In this review, we examine recent work pertaining to microbial community dynamics and succession during vertebrate (human and other mammals) decomposition in terrestrial ecosystems, through the lens of a microbial succession ecological framework. From this perspective, we describe three major microbial microhabitats (internal, external, and soil) in terms of their unique successional trajectories and identify three major knowledge gaps that remain to be addressed.
... The multiplication of microorganisms reaches a peak density of 10 6 microorganisms per milliliter of body fluid or per gram of body tissues within 24 to 30 h of death [9]. Some thanatomicrobiome studies have reported the occurrence of opportunistic bacteria such as Enterobacter sp., E. coli, Pseudomonas sp., Rothia sp., and Streptococcus sp. in cadavers [5,10,11]. Potential Atmosphere 2022, 13, 1281 2 of 16 pathogens have been consistently recovered from bodily fluids and/or aspirates of bodies that were previously certified to have died of causes other than an infectious disease [9,12]. ...
... Keane et al. contend that potential exposure to bioaerosols exists as soon as bodies are manipulated [25]. Research has been carried out to describe the thanatomicrobiome, the disinfection efficiency of the liquids used for embalming, and occupationally-acquired infections by means of questionnaires and surveys [5,10,14,17,20,[26][27][28], but very few studies have taken measurements of exposure during cadaver embalming [29]. The objective of this study was to evaluate the exposure of embalmers to bioaerosols by determining in real time the concentrations and size distribution of airborne fluorescent and non-fluorescent particles, in addition to characterizing the airborne culturable microorganisms in the ambient air. ...
... Keane et al. contend that potential exposure to bioaerosols ex as soon as bodies are manipulated [25]. Research has been carried out to describe the th atomicrobiome, the disinfection efficiency of the liquids used for embalming, and oc pationally-acquired infections by means of questionnaires and surveys [5,10,14,17,20,28], but very few studies have taken measurements of exposure during cadaver emba ing [29]. The objective of this study was to evaluate the exposure of embalmers to bioa osols by determining in real time the concentrations and size distribution of airborne orescent and non-fluorescent particles, in addition to characterizing the airborne cultu ble microorganisms in the ambient air. ...
Embalmers are exposed to many pathogens present in bodily fluids. However, the risk posed by these pathogens has yet to be defined in terms of the nature of the hazard and the intensity of the exposure. The objective of this project was to monitor the exposure of embalmers to biological particles in real time and to characterize the microbiota found in the air during embalming activities in three thanatopraxy laboratories. An innovative approach, using a laser-induced fluorescence aerosol spectrometer (WIBS-NEO), made it possible to measure the concentrations and particle size distributions of the aerosols (biological and non-biological) emitted during embalming. At the same time, an Andersen impactor was used to sample the culturable microbiota present in the air and perform its characterization. The preferential aerosolization of the biological (fluorescent) fraction during embalming procedures, which was compared to the non-biological (non-fluorescent) fraction, showed that most of the tasks performed by the embalmer are likely to lead to microbial exposure via bioaerosols. The concentrations measured represented the equivalent of 2000 to 10,000 biological particles inhaled per minute. Although Mycobacterium tuberculosis was not identified in the air during this study, the presence of Streptococcus pneumoniae in some of the samples demonstrated that if a pathogen is present in the lungs of the deceased it can be aerosolized and inhaled by the embalmers. The size distribution showed that embalmers are exposed to a high proportion of small particles in the aerosols produced during their work. Thus, the respirable/total ratios calculated are between 58% and 78%. Finally, the detection of airborne Enterobacter, Serratia, Leclercia, and Hafnia tended to demonstrate the aerosolization of intestinal bacteria and their possible inhalation or ingestion. Due to the difficulty of identifying the presence of pathogenic agents before embalming, the presence of faecal bacteria in the air, the proximity of the embalmer to the body, and the limitations associated with the dilution of contaminants by general ventilation in the near field, local ventilation must be provided. Otherwise, minimally, a fitted N95-type respirator should be recommended.
... Some studies use a more generalized term for thanatomicrobiome, defining it as the postmortem microbial community of the human body, which involves a successional process where trillions of microbes inhabit, proliferate, and die internally and externally throughout the dead body, resulting in temporal shifts in community composition over time [69][70][71][72]. ...
... Swine are the preferred non-human model as their decomposition is similar to that of humans [81]. Human cadavers used for postmortem microbial studies are donated bodies from willed donation programs to outdoor decomposition research facilities [69,71,[82][83][84][85][86] or autopsied cadavers in a medical examiner/coroner's office [70,72,87,88]. Different studies propose collecting the samples by swabbing the area of study or dissecting the tissue. ...
... Microbial DNA can be collected from samples using classical methods of DNA extraction, such as the phenol/chloroform method. However, when dealing with samples containing soil, soil DNA extraction kits are highly recommended to mitigate PCR inhibition by the humic acid present in the soil [70,71,81,82]. ...
There are several methodologies available to estimate time since death based on different changes that a corpse undergoes after death. However, these methods are imprecise due to the decomposition process being affected by several factors, principally temperature and humidity. Current trends for the determination of the Postmortem Interval (PMI) attempt to estimate the PMI in a quantifiable manner, based on chemical changes on and in the body, summarized in the field of “thanatochemistry”. Although these methodologies have improved PMI estimates, additional research has been developed to increase the accuracy and precision of this determination. As a result, the fields of “thanatobiology” and “thanatomicrobiome” have emerged. Thanatobiology is based on the estimation of the PMI from DNA/RNA degradation, signaling pathways of cell death, and protein analysis. Thanatomicrobiome refers to changes in the bacterial communities as a consequence of the decomposition process. Although both approaches seem to improve PMI estimates, applications of thanatobiology methodologies are more appropriate in the first phases of decomposition, while thanatomicrobiome analyses are applicable in advanced stages. Further research is needed in these new fields in order to establish their applicability in forensic cases. This is a review of the current state-of-the-art methodology in these two subfields.
... Given the limited access to human cadavers, non-criminal research to thoroughly understand the postmortem microbial community is most often carried out on an animal model (i.e. pigs or mice) (Heimesaat et al. 2012;Can et al. 2014;Lawrence et al. 2019). The real challenge, however, is a correct interpretation of the results, requiring not only assessment by an experienced microbiologist, but also as much comparative data as possible, collected during research or criminal investigations e.g., medical history of the deceased, toxicological analysis, details about the location of the body or environmental conditions (Javan et al. 2019;Ventura Spagnolo et al. 2019, Wójcik et al. 2021. ...
... Their importance in forensic analysis is significant, especially when trying to define the PMI (post-mortem interval), which is the time from death until the discovery of the body. Some human organs e.g., the brain, liver, spleen or heart, are considered to be more sterile than the rest, so any differences in the diversity and abundance of microorganisms observed in these organs after death could indicate the occurrence of migration (Can et al. 2014;Javan et al. 2016a;Zhou and Bian 2018). Moreover, the advancement of these changes could be the reliable marker for determining the time of death of a given individual. ...
... Another issue is the presence of a pathogenic strain in the tested sample. If the presence of a given pathogen was not the direct cause of its death, it may indicate, for example, latent bacteremia (Can et al. 2014;Ventura Spagnolo et al. 2019). The presence of bacteria such as Escherichia coli and other Enterobacteriaceae, Staphylococcus aureus, Streptococcus pneumoniae, and some yeast-like fungi e.g., Candida albicans or Cryptococcus neoformans, always indicates an infection or could be contamination from skin or intestine. ...
Forensic microbiology, also known as the microbiology of death, is an emerging branch of science that is still underused in criminal investigations. Some of the cases might be difficult to solve with commonly-used forensic methods, and then they become an operational field for microbiological and mycological analysis. The aim of our review is to present significant achievements of selected studies on the thanatomicrobiome (microorganisms found in the body, organs and fluids after death) and epinecrotic community (microorganisms found on decaying corpses) that can be used in forensic sciences. Research carried out as a part of the forensic microbiology deals with the thanatomicrobiome and the necrobiome - communities of microorganisms that live inside and outside of a putrefying corpse. Change of species composition observed in each community is a valuable feature that gives a lot of information related to the crime. It is mainly used in the estimation of post-mortem interval (PMI). In some criminal investigations, such noticeable changes in the microbiome and mycobiome can determine the cause or the actual place of death. The microbial traces found at the crime scene can also provide clear evidence of guilt. Nowadays, identification of microorganisms isolated from the body or environment is based on metagenome analysis and 16S rRNA gene amplicon-based sequencing for bacteria and ITS rRNA gene amplicon-based sequencing for fungi. Cultivation methods are still in use and seem to be more accurate; however, they require much more time to achieve a final result, which is an unwanted feature in any criminal investigation.
... Several studies have investigated which microbial communities were prevalent in specific organs during decomposition. The most abundant taxa of microbial communities according to the organ examined are Firmicutes in the brain, heart and spleen [12,24,30]; Firmicutes and Proteobacteria in the liver [12,13,30]; Bacteroidetes, Firmicutes and Pro-teobacteria in the oral cavity [31]; Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes and Proteobacteria in the bones [32]. According to another study, the predominant phyla in the post-mortem microbiota are Firmicutes and Proteobacteria [33]. ...
... Several studies have investigated which microbial communities were prevalent in specific organs during decomposition. The most abundant taxa of microbial communities according to the organ examined are Firmicutes in the brain, heart and spleen [12,24,30]; Firmicutes and Proteobacteria in the liver [12,13,30]; Bacteroidetes, Firmicutes and Pro-teobacteria in the oral cavity [31]; Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes and Proteobacteria in the bones [32]. According to another study, the predominant phyla in the post-mortem microbiota are Firmicutes and Proteobacteria [33]. ...
Forensic microbiology is a relatively new discipline, born in part thanks to the development of advanced methodologies for the detection, identification and characterization of microorganisms, and also in relation to the growing impact of infectious diseases of iatrogenic origin. Indeed, the increased application of medical practices, such as transplants, which require immunosuppressive treatments, and the growing demand for prosthetic installations, associated with an increasing threat of antimicrobial resistance, have led to a rise in the number of infections of iatrogenic origin, which entails important medico-legal issues. On the other hand, the possibility of detecting minimal amounts of microorganisms, even in the form of residual traces (e.g., their nucleic acids), and of obtaining gene and genomic sequences at contained costs, has made it possible to ask new questions of whether cases of death or illness might have a microbiological origin, with the possibility of also tracing the origin of the microorganisms involved and reconstructing the chain of contagion. In addition to the more obvious applications, such as those mentioned above related to the origin of iatrogenic infections, or to possible cases of infections not properly diagnosed and treated, a less obvious application of forensic microbiology concerns its use in cases of violence or violent death, where the characterization of the microorganisms can contribute to the reconstruction of the case. Finally, paleomicrobiology, e.g., the reconstruction and characterization of microorganisms in historical or even archaeological remnants, can be considered as a sister discipline of forensic microbiology. In this article, we will review these different aspects and applications of forensic microbiology.
... The intended application of the forensic microbiome investigation is represented by the postmortem interval (PMI) estimation (Metcalf et al., 2013;Belk et al., 2018). Most of the studies related to the postmortem microbial diversity from human bodies were performed in the USA, primarily in Michigan (Pechal et al., 2018), Texas (Hyde et al., 2013), Alabama (Can et al., 2014), and Tennessee (Damann et al., 2015;Hauther et al., 2015;Johnson et al., 2016;Adserias-Garriga et al., 2017;DeBruyn and Hauther, 2017), while scarce studies were performed in Europe. To emphasize bacteria as "postmortem clocks, " several studies (Hyde et al., 2013;Bucheli and Lynne, 2016) addressed the species/ community's identification and their succession. ...
... Among shared taxa, Clostridium spp. was observed during the first 72 h postmortem, up to the entire duration of decomposition (Plueckhahn, 1986;Javan et al., 2016Javan et al., , 2019, similar to our study, where Clostridium sensu stricto was predominant for the face samples. However, the representatives of this genus have been observed mostly in organ samples toward the end of the bloat stage (Hyde et al., 2013(Hyde et al., , 2015Can et al., 2014;DeBruyn and Hauther, 2017). ...
Introduction
Forensic microbiome studies expanded during the last decade, aiming to identify putative bacterial biomarkers to be used for the postmortem interval (PMI) estimation. Bacterial diversity and dynamics during decomposition are influenced by each individual’s micro and macroenvironment, ante and postmortem conditions, varying across body sites and time. The skin, the largest organ of the human body, hosts a diverse microbial diversity, representing the first line of defense of a living individual. Targeting the investigation of the postmortem skin microbiome could help understanding the role of microbes during decomposition, and association with the ante and postmortem conditions.
Methods
The current study aimed to identify the postmortem skin microbiome signatures associated with eight human bodies, received at the Institute of Legal Medicine Iasi, Romania, during April and May 2021. A total of 162 samples (including triplicate) representing face and hands skin microbiome were investigated via Illumina MiSeq, upon arrival at the morgue (T0) and after 12 hours (T1).
Results
The taxonomic characteristics of the skin microbiota varied across different body sites. However, there were no significant differences in taxonomic profiles between collection time, T0 and T1, except for some dynamic changes in the abundance of dominant bacteria. Moreover, different microbial signatures have been associated with a specific cause of death, such as cardiovascular disease, while an elevated blood alcohol level could be associated with a decrease in bacterial richness and diversity.
Discussion
The places where the bodies were discovered seemed to play an important role in explaining the bacterial diversity composition. This study shows promising results towards finding common postmortem bacterial signatures associated with human cadavers within the first 12h at the morgue.
... The spread of bacteria to different areas of the body occurs by microbial invasion of the capillaries of the lymphatic and vascular system (Paczkowski and Schultz, 2011) and by invasion of the mucus membranes in the respiratory system (Gill et al., 1976) [6] . The reason for studying microorganisms associated with internal organ tissues is that they are less affected by environmental conditions than those associated with external organ tissues such as the skin or oral mucosa, and they are not directly affected by the proliferation of gut microorganisms that occurs rapidly after human death (Ismail et al., 2014) [21] . In forensic science, it is important to study these microorganisms because the presence/absence and/or abundance of certain bacteria might be indicative of the elapsed time since death (i.e., the post-mortem interval, PMI) as demonstrated in mouse and swine studies (Metcalf et al., 2013, Pechal et al., 2013). ...
... In forensic science, it is important to study these microorganisms because the presence/absence and/or abundance of certain bacteria might be indicative of the elapsed time since death (i.e., the post-mortem interval, PMI) as demonstrated in mouse and swine studies (Metcalf et al., 2013, Pechal et al., 2013). In autopsy microbiology, this information might be important to confirm a suspected antemortem infectionparticularly when the cause of death is unknown (Riedel, 2014) [21] . The amount of ethanol formed during the post-mortem interval is considered to depend on the species of microorganisms present, the availability of substrates, the ante-mortem conditions of the deceased and the storage condition of the body prior to collection of specimens for toxicological analysis (Vassiliki et al., 2008). ...
Human microbiome refers to all microscopic lifeforms such as bacteria, viruses, algae, and fungi inhabiting the human body. Forensic microbiology involves the identification of microorganisms based on post-mortem interval and their distribution in different parts of the body which helps in individual identification, cause of death determination, geolocation determination of where the corpse was possibly found and body fluid identification. Microbial forensics is used to study the transmission of microbes and diseases caused by microbes in sexual assault cases, bio crimes or any other forms of criminal cases. Advancements in molecular biology and genetics have led to the development of analytical instruments and techniques that help in better analysis of microbial samples and its metabolites. Thanato-microbiology refers to the study of microflora residing on body surfaces which is also an evolving field of study in forensic microbiology which mainly helps in distinguishing one individual from another based on the unique microflora inhabiting their body.
... Given the substantial pathological commonalities of cause of death from or with SARS-CoV-2, we investigated the possibility of a microbial fingerprint in organs from SARS-CoV-2 deaths. A human OPEN ACCESS EDITED BY Tatiana Venkova, Fox Chase Cancer Center, United States corpse subsists as a specialized disturbance habitat that selects for a distinct thanatomicrobiome structure capable of decomposing the host depending on the cause of death and abiotic and biotic factors surrounding the death (Can et al., 2014;Javan et al., 2016;Kaszubinski et al., 2020). SARS-CoV-2 binds to angiotensinconverting enzyme 2 (ACE2) receptors present on the surface of various cells in the body and can negatively affect essentially all organs of the host. ...
... Genomic DNA was extracted from internal organs by physical disruption using the phenol-chloroform method, which is specifically optimized for recovery of microbial DNA from low-yield samples (Can et al., 2014). The quality and quantity of DNA was determined by spectrophotometry (NanoDrop ™ ). ...
Introduction: The fact that SARS-CoV-2, the coronavirus that caused COVID-19, can translocate within days of infection to the brain and heart and that the virus can survive for months is well established. However, studies have not investigated the crosstalk between the brain, heart, and lungs regarding microbiota that simultaneously co-inhabit these organs during COVID-19 illness and subsequent death. Given the significant overlap of cause of death from or with SARS-CoV-2, we investigated the possibility of a microbial fingerprint regarding COVID-19 death.
Methods: In the current study, the 16S rRNA V4 region was amplified and sequenced from 20 COVID-19-positive and 20 non-COVID-19 cases. Nonparametric statistics were used to determine the resulting microbiota profile and its association with cadaver characteristics. When comparing non-COVID-19 infected tissues versus those infected by COVID-19, there is statistical differences ( p < 0.05) between organs from the infected group only.
Results: When comparing the three organs, microbial richness was significantly higher in non-COVID-19-infected tissues than infected. Unifrac distance metrics showed more variance between control and COVID-19 groups in weighted analysis than unweighted; both were statistically different. Unweighted Bray-Curtis principal coordinate analyses revealed a near distinct two-community structure: one for the control and the other for the infected group. Both unweighted and weighted Bray-Curtis showed statistical differences. Deblur analyses demonstrated Firmicutes in all organs from both groups.
Discussion: Data obtained from these studies facilitated the defining of microbiome signatures in COVID-19 decedents that could be identified as taxonomic biomarkers effective for predicting the occurrence, the co-infections involved in its dysbiosis, and the evolution of the virus.
... Forensic microbiology first gained global recognition in 2001 as a result of the Bacillus anthracis attacks through the USA postal service. In previous studies on forensic microbiology, forensic microbiological technologies were not specifically described, except for agar cultures for bacteria and fungi combined with PCR for certain microorganisms (Aoyagi et al., 2009;Huys et al., 2012;Uchiyama et al., 2012;Tuomisto et al., 2013;Can et al., 2014;Hauther et al., 2015;Yu et al., 2021a,b). To date, though more than 2,460 different species are presented in The Ribosomal Database Project stores (Maidak et al., 2000), most microbes in the environment have not been described and accessed for biotechnology. ...
... RF models are widely used for PMI estimation and postmortem submersion interval (PMSI) diagnosis (Metcalf et al., 2013;Pechal et al., 2014;Cartozzo et al., 2021;Randall et al., 2021;Zhang et al., 2021;Kaszubinski et al., 2022;Zhao et al., 2022). In 2013, Metcalf et al. (2013 first proposed the concept of a "microbial clock" to infer PMI by setting up regression models using an RF. ...
Forensic microbiology has been widely used in the diagnosis of causes and manner of death, identification of individuals, detection of crime locations, and estimation of postmortem interval. However, the traditional method, microbial culture, has low efficiency, high consumption, and a low degree of quantitative analysis. With the development of high-throughput sequencing technology, advanced bioinformatics, and fast-evolving artificial intelligence, numerous machine learning models, such as RF, SVM, ANN, DNN, regression, PLS, ANOSIM, and ANOVA, have been established with the advancement of the microbiome and metagenomic studies. Recently, deep learning models, including the convolutional neural network (CNN) model and CNN-derived models, improve the accuracy of forensic prognosis using object detection techniques in microorganism image analysis. This review summarizes the application and development of forensic microbiology, as well as the research progress of machine learning (ML) and deep learning (DL) based on microbial genome sequencing and microbial images, and provided a future outlook on forensic microbiology.
... After the host has died, the thought of the microbiome composition functioning as a biomarker has been utilized, and it is especially significant in the field of forensic sciences. (Can et al., 2014) .We have sought a systematic overview of the potential applicability of the Post-mortem Microbiome in forensic medicine and Forensic Microbiology, emphasizing on the various microbial marker profiles, and their robustness as well as limits, with an insistence on the TSD, PMI , manner of death and personal identification. The assessment of TSD is critical in creating relevant information about the time of death, which is required information to be submitted in a court of law. ...
... The intrinsic Post-Mortem Microbiome is also important would not be harmed by gut-related microorganisms that continue to thrive after death. (Can et al., 2014). The gut microbial community is also being looked into as a viable site of microbial activities changes.(DeBruyn ...
... Furthermore, some differences in trends have been related to various postmortem factors such as the cause of death, the PMI, or the location of death [22,33,34]. Also, antemortem factors due to intra personal differences such as health condition, origin, lifestyle, gender, weight or diet, can influence the thanatomicrobiome responsible for an alteration in the VOC generation [35]. ...
... The scientific community agreed that this untimely use of odour evidence in a court trial did have a negative impact on the scientific evolution of GC-based odour analysis for forensic applications [35]. Nevertheless, a number of research groups has invested time and resources to advance GCÂGC for decomposition odour analysis and other forensic applications resulting in several promising examples that have shown that GCÂGC has a great potential to assist the requests of forensic questions. ...
In forensic science, the emission of odours from objects or biological matrices is exploited for different purposes. For example, the monitoring of odours via biological or analytical detectors is used in thanatochemistry, the chemistry of death. The analysis of decomposition odour can be explored to support the localization of a missing body, a scenario encountered in urban search and rescue operations. A better understanding of the formation and evolution of decomposition odour is also of high interest to human remains detection canine handlers to improve training practices and chose appropriate training aids. Next to thanatochemistry, many other types of evidence evaluation benefit from the characterization of the volatile profile including the analysis of fire debris, chemical threat agents, explosives, and drugs.
From a chemical point of view, an odour represents a complex mixture of gaseous molecules and its characterization demands for a powerful analytical technique. Especailly, in non-targeted analysis, the separation power provided by one-dimensional (1D) gas chromatography (GC) can be surpassed. Thus, a better insight is usually achieved using a multidimensional technique, such as comprehensive two-dimensional gas chromatography (GC×GC). This chapter focuses on scientific articles published between 2015 and 2020 reporting on the use of GC×GC for odour characterization in the context of forensic science. The main points are decomposition odour, volatolomic applications for profiling of human scent and illegal trade goods such as wildlife parts. Furthermore, the investigation of volatile traces of drugs and ignitable liquids in the context of arson investigations is addressed in detail. For each section, the length is proportional to the number of publications from the literature review.
... The succession of the human microbiome after death as well as associated soil microbial communities (in the case of outdoor crime scenes with bodies lying on the ground) has proven to be a suitable biomarker for the PMI estimation of human cadavers. Various studies have characterized the microbiome composition at different body sites during the decay process, including spleen, liver, brain, heart, blood, bones, gut, skin and oral cavity [11,[26][27][28][29][30][31][32], with error rates as low as 1.7 days when analysing the first 25 days of decomposition [11]. However, some of these studies have reported confounding effects of the individual [31], sex [26] and abiotic factors (e.g. ...
... However, some of these studies have reported confounding effects of the individual [31], sex [26] and abiotic factors (e.g. ambient temperature, solar irradiance) [27,31] on the microbiome succession, which can contribute to misleading PMI estimations and, hence, should be further investigated. ...
Over the last few years, advances in massively parallel sequencing technologies (also referred to next generation sequencing) and bioinformatics analysis tools have boosted our knowledge on the human microbiome. Such insights have brought new perspectives and possibilities to apply human microbiome analysis in many areas, particularly in medicine. In the forensic field, the use of microbial DNA obtained from human materials is still in its infancy but has been suggested as a potential alternative in situations when other human (non-microbial) approaches present limitations. More specifically, DNA analysis of a wide variety of microorganisms that live in and on the human body offers promises to answer various forensically relevant questions, such as post-mortem interval estimation, individual identification, and tissue/body fluid identification, among others. However, human microbiome analysis currently faces significant challenges that need to be considered and overcome via future forensically oriented human microbiome research to provide the necessary solutions. In this perspective article, we discuss the most relevant biological, technical and data-related issues and propose future solutions that will pave the way towards the integration of human microbiome analysis in the forensic toolkit.
... To build a robust model with low error rates, a time series of samples is required to capture the microbial succession that occurs at the sample location during decomposition (Deel et al., 2019). The results of both Hyde (2013Hyde ( , 2015 and Can (2014) studies confirmed the efficacy of swabbing methods similar to those perfected by HMP protocols. According to thanatomicrobiome studies, the results revealed that the swabbing method provided a higher microbial diversity. ...
... Thanatomicrobiome studies assume that as a human body decays, time-dependent changes in the microbial communities within its internal organs also occur in a wellpatterned predictive manner suggestive of the time since death (Can et al., 2014). The information contained in the thanatomicrobiome not only has potential to improve PMI estimates, but could also provide clues for the decomposition location of the cadaver; both of which are of great interest to the forensic death investigators. ...
... Bodies found at crime scenes are often only in their early decomposition stage, which is hard to observe due to fast changes occurring during this time for the body. Can, Javan, Pozhitkov, and Noble (2014) conducted research on microbiological populations in cadavers and observed that lactobacillus, a species of anaerobic bacteria, was present in cadavers with shorter postmortem intervals, which suggests that observing bacteria populations of specific species could help with determining the postmortem interval in human cadavers. However, another bacteria species, clostridium, was present on multiple cadavers at different decomposition times, which means that the bacteria populations of different species need to be heavily studied in order to prevent mistakes (Can et al., 2014). ...
... Can, Javan, Pozhitkov, and Noble (2014) conducted research on microbiological populations in cadavers and observed that lactobacillus, a species of anaerobic bacteria, was present in cadavers with shorter postmortem intervals, which suggests that observing bacteria populations of specific species could help with determining the postmortem interval in human cadavers. However, another bacteria species, clostridium, was present on multiple cadavers at different decomposition times, which means that the bacteria populations of different species need to be heavily studied in order to prevent mistakes (Can et al., 2014). Javan and colleagues (2016) also suggest creating a catalogue of bacteria that records the presence and migrations of different bacterial species in cadavers while accounting for temperature and the body part location being studied. ...
... Each has diverse bacterial populations, which suggests the potential of microbiomes in criminal investigations to estimate the postmortem interval. In the future, it may be possible to investigate different microbiome communities in terms of decomposition to determine the actual time since death in addition to gender-specific microbiomes [7,26]. Similar and repeatable microbiomes influence mammalian decomposition in various hosts and environments. ...
Recent breakthroughs in forensic sciences, bioinformatics and next-generation sequencing technologies have broadened the application of microbiome analysis as an upcoming forensic tool. Studying the variation of the microbial flora and their profile, as well as the interactions among microorganisms, hosts, and the environment, are recent topics in microbiome research worldwide. Such novel microbiome applications have created a wide range of additional opportunities for the advancement of the forensic science. There are many modern forensic uses for the microbiome, such as postmortem identification, geolocation inference, and post-mortem interval prediction.
... The microorganisms studied in the thanatomicrobiome are internal organs such as the brain, spleen, heart, intestines, liver, and others. 5,6 Among these organs, the highest microbial diversity lies in the liver according to Can et al. 17 Another community included in the post-mortem microbiome of humans is the epinecrotic microbial community. Microorganisms in this community are protists, prokaryotes, fungi, and other microeukaryotes that live on the surface of the corpse. ...
Various methods for estimating post-mortem (PMI) have been investigated such as rigor mortis, livor mortis, molecular, chemical, and forensic entomology (insect) methods. However, these methods have weaknesses, therefore researchers are looking for more accurate methods in determining the time of death of a person. Molecular methods are unreliable due to the degradation of DNA, RNA, and proteins in corpses over time. In different parts of the body, the amount of diversity of microbes will be different. The oral cavity is the most abundant microbial area among other parts of the body due to its continuous exposure to the respiratory and digestive systems. This area has great potential in estimating time intervals of death because of its ease of access and the types of microbiomes that predominate at any given time. Therefore, this narrative review was conducted to describe studies that used oral microbiota communities to estimate post-mortem intervals. Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes are the dominant microbial types found in corpses. Firmicutes became one of the dominant bacterial phyla in the early stages of decomposition. Actinobacteria were found to decrease as PMI increased. Studies have shown that the oral microbiome has excellent potential as a parameter to determine the post-mortem interval. However, further research is needed with more complex environmental conditions such as different humidity and temperature. In addition, further research requires more samples of human remains to obtain more valid results. Keywords: oral microbiome; post-mortem interval; human identification; forensic odontology
... Conversely, bacteria in the Enterobacteriaceae and Lactobacillaceae families dominated uterus samples. Overall, there was a substantially lower abundance of the Clostridiaceae family, which contrasts to a previous study (37) where the abundance was higher in faster decaying internal organs such as the liver and spleen (38,39). ...
Significant knowledge gaps exist regarding the responses of cells, tissues, and organs to organismal death. Examining the survival mechanisms influenced by metabolism and environment, this research has the potential to transform regenerative medicine, redefine legal death, and provide insights into life's physiological limits, paralleling inquiries in embryogenesis.
... The microbiome of death is defined as microbial succession in the internal organs (e.g., brain, heart, liver, spleen) of decomposing humans and can provide physical and molecular evidence concerning interactions between putrefactive microorganisms and their mammalian hosts (Hyde et al., 2013(Hyde et al., , 2015Can et al., 2014;Damann et al., 2015;Javan et al., 2016aJavan et al., ,b, 2017Metcalf et al., 2016;Pechal et al., 2018;Lutz et al., 2020; Figure 2). It is a longheld belief that mammalian internal organs are sterile in living hosts (Ford, 1901;Stewart, 2012). ...
Human death is a complex, time-governed phenomenon that leads to the irreversible cessation of all bodily functions. Recent molecular and genetic studies have revealed remarkable experimental evidence of genetically programmed cellular death characterized by several physiological processes; however, the basic physiological function that occurs during the immediate postmortem period remains inadequately described. There is a paucity of knowledge connecting necrotic pathologies occurring in human organ tissues to complete functional loss of the human organism. Cells, tissues, organs, and organ systems show a range of differential resilience and endurance responses that occur during organismal death. Intriguingly, a persistent ambiguity in the study of postmortem physiological systems is the determination of the trajectory of a complex multicellular human body, far from life-sustaining homeostasis, following the gradual or sudden expiry of its regulatory systems. Recent groundbreaking investigations have resulted in a paradigm shift in understanding the cell biology and physiology of death. Two significant findings are that (i) most cells in the human body are microbial, and (ii) microbial cell abundance significantly increases after death. By addressing the physiological as well as the microbiological aspects of death, future investigations are poised to reveal innovative insights into the enigmatic biological activities associated with death and human decomposition. Understanding the elaborate crosstalk of abiotic and biotic factors in the context of death has implications for scientific discoveries important to informing translational knowledge regarding the transition from living to the non-living. There are important and practical needs for a transformative reestablishment of accepted models of biological death (i.e., artificial intelligence, AI) for more precise determinations of when the regulatory mechanisms for homeostasis of a living individual have ceased. In this review, we summarize mechanisms of physiological, genetic, and microbiological processes that define the biological changes and pathways associated with human organismal death and decomposition.
... Microbial findings Estimated PMI [47] Blood, brain, heart, liver and spleen Clostridium sp. in heart, liver, spleen and Lactobacillus in blood were prominent bacterial species observed. of various microbial species [40,41]. Researchers can determine how the relative abundance of different taxa has changed over time by analyzing the sequencing data derived from time-series samples [10,38,39]. ...
Cadaver decomposition is a natural phenomenon intimately affected by numerous organisms such as insects, bacteria etc., where they use the decaying body as their nutrition source. These organisms can be utilized in forensic science to estimate the Post-mortem Interval. Forensic entomology is one of the popular approaches where successive colonization of insects on cadaver is studied to estimate PMI. However, sometime this method does not provide consistent results due to lack of insect activities during cold environment conditions or when crime scene is indoor. Recently, researchers have noted that microbiomes have shown predictable and clockwise successional patterns on decomposing cadavers and suggested this could be utilized to estimate PMI when this approach is etched with other established methods. This chapter summarizes the utility of microbial profiling in medico-legal investigations.
... Moreover, for a better understanding of how DNA and bone degrade in different environmental conditions, experiments were performed on different types of animal and human bones (Bell et al. 1996;Cartozzo et al. 2021;Deel et al. 2021;Emmons et al. 2019;Emmons et al. 2022;Kazarina et al. 2019;Reeb et al. 2011;White and Booth 2014). In terms of microbial activity, such studies showed a shift in the dominant bacteria involved in decay from endogenous obligate and facultatively anaerobic organisms of the Bacteroidetes and Firmicutes in the early postmortem period (Can et al. 2014;Damann and Jans 2017;Hyde et al. 2013;Metcalf et al. 2013;Pechal et al. 2014) to Proteobacteria and Actinobacteria. Thus, over time, a change in the microbial community of the bones could be observed, becoming more alike to the corresponding sediment (Cobaugh et al. 2015;Damann et al. 2015;Damann and Jans 2017). ...
... Estimation of PMI is a complex forensic assessment, and differential flux in the succession of colonizing microbial communities in major organs has been studied in animal models of pigs and mice 126,127 to improve the accuracy of PMI estimation. Such sequences of exogenous and endogenous bacterial species have been shown to be useful for PMI estimation 128 and to allow assignment to a specific time interval. ...
In contrast to other forensic disciplines, forensic microbiology is still too often considered a "side activity" and is not able to make a real and concrete contribution to forensic investigations. Indeed, the various application aspects of this discipline still remain a niche activity and, as a result, microbiological investigations are often omitted or only approximated, in part due to poor report in the literature. However, in certain situations, forensic microbiology can prove to be extremely effective, if not crucial, when all other disciplines fail. Precisely because microorganisms can represent forensic evidence, in this narrative review all the major pathological forensic applications described in the literature have been presented. The goal of our review is to highlight the versatility and transversality of microbiology in forensic science and to provide a comprehensive source of literature to refer to when needed.
... Our data indicated that greater differences among water samples provide more useful information for inferring the drowning site, but this method still has limitations. The relative abundance of microorganisms in blood and internal organs changes dynamically in different postmortem intervals (Can et al., 2014;Javan et al., 2016), which can potentially affect the accuracy of inferring drowning sites and requires further evaluation. Furthermore, it is important to note that our current research was primary research conducted on animals, which have a similar microbiome due to their consistent biological background. ...
Diagnosing the drowning site is a major challenge in forensic practice, particularly when corpses are recovered from flowing rivers. Recently, forensic experts have focused on aquatic microorganisms, including bacteria, which can enter the bloodstream during drowning and may proliferate in corpses. The emergence of 16S ribosomal RNA gene (16S rDNA) amplicon sequencing has provided a new method for analyzing bacterial composition and has facilitated the development of forensic microbiology. We propose that 16S rDNA amplicon sequencing could be a useful tool for inferring drowning sites. Our study found significant differences in bacterial composition in different regions of the Guangzhou section of the Pearl River, which led to differences in bacteria of drowned rabbit lungs at different drowning sites. Using the genus level of bacteria in the lung tissue of drowned rabbits, we constructed a random forest model that accurately predicted the drowning site in a test set with 100% accuracy. Furthermore, we discovered that bacterial species endemic to the water were not always present in the corresponding drowned lung tissue. Our findings demonstrate the potential of a random forest model based on bacterial genus and composition in drowned lung tissues for inferring drowning sites.
... Yet, far less is understood about microbial communities, their actions and interactions, in applied settings, such as crime scenes. The specific attributes of certain microbes can be of great investigative value as trace evidence in criminal cases [24][25][26][27][28]. Several studies have provided indications that microbes can help to determine both cause of death and time since death [19,[29][30][31], and may contribute to the identification of victim remains [25,32], through the examination of their diversity, abundance, and succession over time [30,[33][34][35]; bearing in mind the dependence of any results and conclusions on local environmental conditions. It has been proven that the microbial 'signature' present in the human skin [36,37] can be transferred along with the convoluted mixture of nutrients in fingerprints [38,39]; hence, changes in the relative composition of microbes over time once transferred onto a surface could similarly provide valuable data to estimate TSD. ...
Interest in the human microbiome has grown in recent years because of increasing applications to biomedicine and forensic science. However, the potential for dating evidence at a crime scene based upon time-dependent changes in microbial signatures has not been established, despite a relatively straightforward scientific process for isolating the microbiome. We hypothesize that modifications in microbial diversity, abundance, and succession can provide estimates of the time a surface was touched for investigative purposes. In this proof-of-concept research, the sequencing and analysis of the 16 S rRNA gene from microbes present in fresh and aged latent fingerprints deposited by three donors with pre- and post-washed hands is reported. The stability of major microbial phyla is confirmed while the dynamics of less abundant groups is described up to 21 days post-deposition. Most importantly, a phylum is suggested as the source for possible biological markers to date fingerprints: Deinococcus-Thermus.
... Post-mortem microbiomes have been studied intensively since the advent of next generation sequencing in the context of forensic science, medicine, and decomposition ecology since they are informative of a variety of factors associated with death investigations, the physiological states of the previously living, and ecosystem functions (Metcalf et al., 2013;Pechal et al., 2014;Cobaugh et al., 2015;Singh et al., 2018;Kaszubinski et al., 2020). There is experimental evidence indicating that microbial data can provide information associated with post-mortem intervals (PMI), locations of death, and causes of death (Can et al., 2014;Finley et al., 2015;Pechal et al., 2018;Zhang et al., 2019). In post-mortem microbiome work, the general concept is that a decedent is a microbial ecosystem that will undergo a predictable succession of community members over time. ...
Background
Post-mortem microbial communities are increasingly investigated as proxy evidence for a variety of factors of interest in forensic science. The reported predictive power of the microbial community to determine aspects of the individual’s post-mortem history (e.g., the post-mortem interval) varies substantially among published research. This observed variation is partially driven by the local environment or the individual themselves. In the current study, we investigated the impact of BMI, sex, insect activity, season, repeat sampling, decomposition time, and temperature on the microbial community sampled from donated human remains in San Marcos, TX using a high-throughput gene-fragment metabarcoding approach.
Materials and methods
In the current study, we investigated the impact of BMI, sex, insect activity, season, repeat sampling, decomposition time, and temperature on the microbial community sampled from donated human remains in San Marcos, TX using a high-throughput gene-fragment metabarcoding approach.
Results
We found that season, temperature at the sampling site, BMI, and sex had a significant effect on the post-mortem microbiome, the presence of insects has a homogenizing influence on the total bacterial community, and that community consistency from repeat sampling decreases as the decomposition process progresses. Moreover, we demonstrate the importance of temperature at the site of sampling on the abundance of important diagnostic taxa.
Conclusion
The results of this study suggest that while the bacterial community or specific bacterial species may prove to be useful for forensic applications, a clearer understanding of the mechanisms underpinning microbial decomposition will greatly increase the utility of microbial evidence in forensic casework.
... For broader application of the findings in forensic science, it would be helpful to develop reliable and robust databases of microbiomes obtained in multiple aquatic environments and seasons. The succession of postmortem microbiota that colonize internal organs (including the gut, brain, liver, spleen, and heart) has proven useful for PMI estimation in terrestrial environments (Can et al., 2014;Javan et al., 2016). The present study provides novel and informative context for better understanding the decomposition processes that submerged corpses undergo, which have important implications for forensic practice. ...
IntroductionBodies recovered from water, especially in the late phase of decomposition, pose difficulties to the investigating authorities. Various methods have been proposed for postmortem submersion interval (PMSI) estimation and drowning identification, but some limitations remain. Many recent studies have proved the value of microbiota succession in viscera for postmortem interval estimation. Nevertheless, the visceral microbiota succession and its application for PMSI estimation and drowning identification require further investigation.Methods
In the current study, mouse drowning and CO2 asphyxia models were developed, and cadavers were immersed in freshwater for 0 to 14 days. Microbial communities in the liver and brain were characterized via 16S rDNA high-throughput sequencing.ResultsOnly livers and brains collected from 5 to 14 days postmortem were qualified for sequencing. There was significant variation between microbiota from liver and brain. Differences in microbiota between the cadavers of mice that had drowned and those only subjected to postmortem submersion decreased over the PMSI. Significant successions in microbial communities were observed among the different subgroups within the late phase of the PMSI in livers and brains. Eighteen taxa in the liver which were mainly related to Clostridium_sensu_stricto and Aeromonas, and 26 taxa in the brain which were mainly belonged to Clostridium_sensu_stricto, Acetobacteroides, and Limnochorda, were selected as potential biomarkers for PMSI estimation based on a random forest algorithm. The PMSI estimation models established yielded accurate prediction results with mean absolute errors ± the standard error of 1.282 ± 0.189 d for the liver and 0.989 ± 0.237 d for the brain.Conclusions
The present study provides novel information on visceral postmortem microbiota succession in corpses submerged in freshwater which sheds new light on PMSI estimation based on the liver and brain in forensic practice.
... Some studies revealed changes of dominant microorganisms in different human organs and blood specimens after death. Bacillus and Lactobacillus predominated in the shortterm after death followed by an exponential decrease with the extension of PMI, while parthenogenic anaerobic bacteria, such as Clostridium, were predominant in the late phase of PMI (Can et al., 2014;Hauther et al., 2015;Javan et al., 2016;DeBruyn and Hauther, 2017). This accounted for the phenomenon of Postmortem Clostridium Effect (PCE) at decomposition stage (Javan et al., 2017). ...
Postmortem interval (PMI) estimation has always been a major challenge in forensic science. Conventional methods for predicting PMI are based on postmortem phenomena, metabolite or biochemical changes, and insect succession. Because postmortem microbial succession follows a certain temporal regularity, the microbiome has been shown to be a potentially effective tool for PMI estimation in the last decade. Recently, artificial intelligence (AI) technologies shed new lights on forensic medicine through analyzing big data, establishing prediction models, assisting in decision-making, etc. With the application of next-generation sequencing (NGS) and AI techniques, it is possible for forensic practitioners to improve the dataset of microbial communities and obtain detailed information on the inventory of specific ecosystems, quantifications of community diversity, descriptions of their ecological function, and even their application in legal medicine. This review describes the postmortem succession of the microbiome in cadavers and their surroundings, and summarizes the application, advantages, problems, and future strategies of AI-based microbiome analysis for PMI estimation.
... DNA extraction was performed using the traditional phenol-chloroform method as previously described [24]. Briefly, 2X TENS (100 mM Tris-HCl [pH 8.0], 40 mM EDTA, 200 mM NaCl, 2% SDS) buffer was added in Lysing Matrix E tubes (MP Biomedicals, Santa Ana, CA). ...
The microbiota gut-brain-axis is a bidirectional circuit that links the neural, endocrine, and immunological systems with gut microbial communities. The gut microbiome plays significant roles in human mind and behavior, specifically pain perception, learning capacity, memory, and temperament. Studies have shown that disruptions in the gut microbiota have been associated with substance use disorders. The interplay of gut microbiota in substance abuse disorders has not been elucidated; however, postmortem microbiome profiles may produce promising avenues for future forensic investigations. The goal of the current study was to determine gut microbiome composition in substance abuse disorder cases using transverse colon tissues of 21 drug overdose versus 19 non-overdose-related cases. We hypothesized that postmortem samples of the same cause of death will reveal similar microbial taxonomic relationships. We compared microbial diversity profiles using amplicon-based sequencing of the 16S rRNA gene V4 hypervariable region. The results demonstrated that the microbial abundance in younger-aged cases were found to have significantly more operational taxonomic units than older cases. Using weighted UniFrac analysis, the influence of substances in overdose cases was found to be a significant factor in determining microbiome similarity. The results also revealed that samples of the same cause of death cluster together, showing a high degree of similarity between samples and a low degree of similarity among samples of different causes of death. In conclusion, our examination of human transverse colon microflora in decomposing remains extends emerging literature on postmortem microbial communities, which will ultimately contribute to advanced knowledge of human putrefaction.
... only in male cadavers [8]. Organ thanatomicrobiome analyses suggest that facultative anaerobes, such as Lactobacillus, predominate in the "short PMI" timeframe, while in a "long PMI" timeframe a predominance of obligate anaerobes like Clostridium is observed [53]. Notably, Firmicutes (e.g., Clostridium, Peptoniphilus, and Bacillus) represent a stable and constant biomarker in microbial communities derived from different body locations [33]. ...
Human DNA samples can remain unaltered for years and preserve important genetic information for forensic investigations. In fact, besides human genetic information, these extracts potentially contain additional valuable information: microbiome signatures. Forensic microbiology is rapidly becoming a significant tool for estimating post-mortem interval (PMI), and establishing cause of death and personal identity. To date, the possibility to recover unaltered microbiome signatures from human DNA extracts has not been proven. This study examines the microbiome signatures within human DNA extracts obtained from six cadavers with different PMIs, which were stored frozen for 5-16 years. Results demonstrated that the microbiome can be co-extracted with human DNA using forensic kits designed to extract the human host's DNA from different tissues and fluids during decomposition. We compared the microbial communities identified in these samples with microbial DNA recovered from two human cadavers donated to the Forensic Anthropology Center at Texas State University (FACTS) during multiple decomposition stages, to examine whether the microbial signatures recovered from "old" (up to 16 years) extracts are consistent with those identified in recently extracted microbial DNA samples. The V4 region of 16 S rRNA gene was amplified and sequenced using Illumina MiSeq for all DNA extracts. The results obtained from the human DNA extracts were compared with each other and with the microbial DNA from the FACTS samples. Overall, we found that the presence of specific microbial taxa depends on the decomposition stage, the type of tissue, and the depositional environment. We found no indications of contamination in the microbial signatures, or any alterations attributable to the long-term frozen storage of the extracts, demonstrating that older human DNA extracts are a reliable source of such microbial signatures. No shared Core Microbiome (CM) was identified amongst the total 18 samples, but we identified certain species in association with the different decomposition stages, offering potential for the use of microbial signatures co-extracted with human DNA samples for PMI estimation in future. Unveiling the new significance of older human DNA extracts brings with it important ethical-legal considerations. Currently, there are no shared legal frameworks governing the long-term storage and use of human DNA extracts obtained from crime scene evidence for additional research purposes. It is therefore important to create common protocols on the storage of biological material collected at
... only in male cadavers [8]. Organ thanatomicrobiome analyses suggest that facultative anaerobes, such as Lactobacillus, predominate in the "short PMI" timeframe, while in a "long PMI" timeframe a predominance of obligate anaerobes like Clostridium is observed [53]. Notably, Firmicutes (e.g., Clostridium, Peptoniphilus, and Bacillus) represent a stable and constant biomarker in microbial communities derived from different body locations [33]. ...
Human DNA samples can remain unaltered for years and preserve important genetic information for forensic investigations. In fact, besides human genetic information, these extracts potentially contain additional valuable information: microbiome signatures. Forensic microbiology is rapidly becoming a significant tool for estimating post-mortem interval (PMI), and establishing cause of death and personal identity. To date, the possibility to recover unaltered microbiome signatures from human DNA extracts has not been proven. This study examines the microbiome signatures within human DNA extracts obtained from six cadavers with different PMIs, which were stored frozen for 5–16 years. Results demonstrated that the microbiome can be co-extracted with human DNA using forensic kits designed to extract the human host’s DNA from different tissues and fluids during decomposition. We compared the microbial communities identified in these samples with microbial DNA recovered from two human cadavers donated to the Forensic Anthropology Center at Texas State University (FACTS) during multiple decomposition stages, to examine whether the microbial signatures recovered from “old” (up to 16 years) extracts are consistent with those identified in recently extracted microbial DNA samples. The V4 region of 16 S rRNA gene was amplified and sequenced using Illumina MiSeq for all DNA extracts. The results obtained from the human DNA extracts were compared with each other and with the microbial DNA from the FACTS samples. Overall, we found that the presence of specific microbial taxa depends on the decomposition stage, the type of tissue, and the depositional environment. We found no indications of contamination in the microbial signatures, or any alterations attributable to the long-term frozen storage of the extracts, demonstrating that older human DNA extracts are a reliable source of such microbial signatures. No shared Core Microbiome (CM) was identified amongst the total 18 samples, but we identified certain species in association with the different decomposition stages, offering potential for the use of microbial signatures co-extracted with human DNA samples for PMI estimation in future. Unveiling the new significance of older human DNA extracts brings with it important ethical-legal considerations. Currently, there are no shared legal frameworks governing the long-term storage and use of human DNA extracts obtained from crime scene evidence for additional research purposes. It is therefore important to create common protocols on the storage of biological material collected at crime scenes. We review existing legislation and guidelines, and identify some important limitations for the further development and application of forensic microbiomics.
... Interestingly, studies on internal organs of mice (Metcalf et al., 2013;Burcham et al., 2019), swine (Carter et al., 2015), and human bodies (Hyde et al., 2013;Can et al., 2014;Hauther et al., 2015) have observed a shift in microbial communities from predominant aerobic microorganisms such as Staphylococcus and Enterobacteriaceae to more facultative anaerobic bacteria such as Proteobacteria, Firmicutes, and Bacteroidetes to obligate anaerobic organisms such as Clostridium, and finally sporeforming microorganisms such as Clostridium and Bacillus (Figure 1; Hyde et al., 2015;Javan et al., 2016bJavan et al., , 2019García et al., 2020). According to the "post-mortem Clostridium effect, " Clostridium species can be considered important drivers of this microbial shift due to their lipolytic enzymes (Janaway et al., 2009), proteolytic functions, and rapid generation time (Javan et al., 2017). ...
Microbial forensics represents a promising tool to strengthen traditional forensic investigative methods and fill related knowledge gaps. Large-scale microbiome studies indicate that microbial fingerprinting can assist forensics in areas such as trace evidence, source tracking, geolocation, and circumstances of death. Nevertheless, the majority of forensic microbiome studies focus on soil and internal organ samples, whereas the microbiome of skin, mouth, and especially vaginal samples that are routinely collected in sexual assault and femicide cases remain underexplored. This review discusses the current and emerging insights into vaginal, skin, and salivary microbiome-modulating factors during life (e.g., lifestyle and health status) and after death (e.g., environmental influences and post-mortem interval) based on next-generation sequencing. We specifically highlight the key aspects of female reproductive tract, skin, and mouth microbiome samples relevant in forensics. To fill the current knowledge gaps, future research should focus on the degree to which the post-mortem succession rate and profiles of vaginal, skin, and saliva microbiota are sensitive to abiotic and biotic factors, presence or absence of oxygen and other gases, and the nutrient richness of the environment. Application of this microbiome-related knowledge could provide valuable complementary data to strengthen forensic cases, for example, to shed light on the circumstances surrounding death with (post-mortem) microbial fingerprinting. Overall, this review synthesizes the present knowledge and aims to provide a framework to adequately comprehend the hurdles and potential application of vaginal, skin, and salivary post-mortem microbiomes in forensic investigations.
... Next, Pr. François-Joseph Lapointe (Faculté des arts et des sciences-Département de sciences biologiques, Montréal, Canada) introduced the promises and challenges of the development of forensic methods based on the analyses of post-mortem microbiome dynamics, and described the imminent use of microbiomes as signatures on a crime-scene [31][32][33][34][35][36][37][38]. Pr. ...
Many separate fields and practices nowadays consider microbes as part of their legitimate focus. Therefore, microbiome studies may act as unexpected unifying forces across very different disciplines. Here, we summarize how microbiomes appear as novel major biological players, offer new artistic frontiers, new uses from medicine to laws, and inspire novel ontologies. We identify several convergent emerging themes across ecosystem studies, microbial and evolutionary ecology, arts, medicine, forensic analyses, law and philosophy of science, as well as some outstanding issues raised by microbiome studies across these disciplines and practices. An ‘epistemic revolution induced by microbiome studies’ seems to be ongoing, characterized by four features: (i) an ecologization of pre-existing concepts within disciplines, (ii) a growing interest in systemic analyses of the investigated or represented phenomena and a greater focus on interactions as their root causes, (iii) the intent to use openly multi-scalar interaction networks as an explanatory framework to investigate phenomena to acknowledge the causal effects of microbiomes, (iv) a reconceptualization of the usual definitions of which individuals are worth considering as an explanans or as an explanandum by a given field, which result in a fifth strong trend, namely (v) a de-anthropocentrification of our perception of the world.
... Denaturing gradient gel electrophoresis (DGGE) is an economical and reliable method used in many laboratories around the world, with the generated data analysed further by calculating appropriate ecological indices for richness, diversity and evenness. To date, most published research on characterisation of soil microbial communities for forensic applications have used classic diversity metrics such as Shannon-Wiener, Simpson index of concentration or those built within software/pipeline packages (Can et al., 2014;Olakanye et al., 2017;Olakanye and Ralebitso-Senior, 2018;Procopio et al., 2019). However, these metrics have known shortcomings and generate results that are not easy to interpret. ...
Introducing animal carbon-source to soil initiates biochemical and microbial processes that lead to its decomposition and recycling, which subsequently cause successional shifts in soil microbial community. To investigate the use of soil microbial community to inform criminal investigation, this study was designed to mimic clandestine graves. It compared the decomposition of stillborn piglets (Sus scrofa domesticus), as human analogues, to oak (Quercus robur) leaf litter and soil-only controls outdoors for 720 days. Environmental and edaphic parameters were monitored and showed soil microbial community alignment with temperature seasonality, which highlighted the importance of this abiotic factor. Denaturing gradient gel electrophoresis (DGGE) data were used to calculate Hill numbers and diversity indices of the bacterial 16S rRNA community did not distinguish mammalian- from plant-based decomposition consistently during the first or second year of the study. In contrast, the fungal 18S rRNA community allowed clear differentiation between different treatments (beta diversity) throughout the 720-day experiment and suggested the moment of the decomposing mammalian skin rupture. 16S rRNA-based NGS facilitated the identification of e.g., Pirellulaceae, Acidobacteria ii1-15_order and Candidatus xiphinematobacter as Year 2 bacterial markers of gravesoil at family, order and species taxonomic levels, respectively, and confirmed the similarity of the calculated Hill diversity metrics with those derived from DGGE profiling. Parallel soil elemental composition was measured by portable X-ray Fluorescence where calcium profiles for the piglet-associated soils were distinct from those without carrion. Also, soil calcium content and PMI correlated positively during the first year then negatively during the second. This study is one of the first to apply a multidisciplinary approach based on molecular and physicochemical analytical techniques to assess decomposition. It highlights the recognised potential of using soil microbial community in forensic investigations and provides a proof-of-concept for the application of a combined molecular and elemental approach to further understand the dynamics of decomposition. In addition, it sets the scene for further research in different conditions based on Hill numbers metrics instead of the classic ecological indices for soil necrobiome richness, diversity and evenness.
... Characteristic microbial succession in the internal sites and organs can thus be utilized to predict PMI. In 11 cases in which the PMI ranged from 20 to 240 h, organ thanatomicrobiome analyses suggested that facultative anaerobes, such as Lactobacillus, predominate in cadavers with short PMI (29.5 h), while cadavers with long PMI (240 h) have a predominance of obligate anaerobes like Clostridium (Can et al., 2014). In a cross-sectional study involving 27 human cadavers from criminal cases with known PMI, statistically significant organ-, sex-, and time-dependent differences were found in the thanatomicribome communities of the brain, heart, liver, spleen, buccal cavity, and blood (Javan et al., 2016b). ...
Forensic medicine has, for a long time, been relying on biochemical, anthropologic, and histopathologic evidences in solving various investigations. However, depending on the method used, lengthy sample processing time, scanty sample, and less sensitivity and accuracy pervade these procedures. Accordingly, newer arenas such as the thanatomicrobiome have come forward to aid in its quandaries; furthermore, the parallel advances in genomic and proteomic techniques have complemented and are still emerging to be used in forensic experiments and investigations. Postmortem interval (PMI) is one of the most important aspects of medico-legal investigations. The current trend in PMI estimation is toward genomic analyses of autopsy samples. Similarly, determination of cause of death, although a domain of medical sciences, is being targeted as the next level of forensic casework. With the current trend in laboratory sciences moving to the discovery of newer disease-specific markers for diagnostic and prognostic purposes, the same is being explored for the determination of the cause of death by using techniques such as Real-Time PCR, DNA micro-array, to Next-Gen Sequencing. Establishing an individual’s biological profile has been done using medicolegal methods and anthropology as well as bar-bodies/Davidson bodies (gender determination); and in cases where the determination of age/gender is a challenge using morphological characteristics; the recent advances in the field of genomics and proteomics have played a significant role, e.g., use of mitochondrial DNA in age estimation and in maternity disputes. The major hurdle forensic medical research faces is the fact that most of the studies are conducted in animal models, which are often difficult to mimic in human and real-time scenarios. Additionally, the high accuracy required in criminal investigations to be used in a court of law as evidence has prevented these results to come out of the labs and be used to the optimum. The current review aims at giving a comprehensive and critical account of the various molecular biology techniques including “thanatogenomics,” currently being utilized in the veritable fields of forensic medicine.
Recent advancements in omics techniques have revolutionised the study of biological systems, enabling the generation of high‐throughput biomolecular data. These innovations have found diverse applications, ranging from personalised medicine to forensic sciences. While the investigation of multiple aspects of cells, tissues or entire organisms through the integration of various omics approaches (such as genomics, epigenomics, metagenomics, transcriptomics, proteomics and metabolomics) has already been established in fields like biomedicine and cancer biology, its full potential in forensic sciences remains only partially explored. In this review, we have presented a comprehensive overview of state‐of‐the‐art analytical platforms employed in omics research, with specific emphasis on their application in the forensic field for the identification of the cadaver and the cause of death. Moreover, we have conducted a critical analysis of the computational integration of omics approaches, and highlighted the latest advancements in employing multi‐omics techniques for forensic investigations.
The human microbiome is vital for maintaining human health and has garnered substantial attention in recent
years, particularly in the context of the coronavirus disease 2019 (COVID-19) outbreak. Studies have underscored significant alterations in the microbiome of COVID-19 patients across various body niches, including the
gut, respiratory tract, oral cavity, skin, and vagina. These changes manifest as shifts in microbiota composition,
characterized by an increase in opportunistic pathogens and a decrease in beneficial commensal bacteria. Such
microbiome transformations may play a pivotal role in influencing the course and severity of COVID-19,
potentially contributing to the inflammatory response. This ongoing relationship between COVID-19 and the
human microbiome serves as a compelling subject of research, underscoring the necessity for further investigations into the underlying mechanisms and their implications for patient health. Additionally, these alterations in the microbiome may have significant ramifications for forensic investigations, given the
microbiome’s potential in establishing individual characteristics. Consequently, changes in the microbiome
could introduce a level of complexity into forensic determinations. As research progresses, a more profound
understanding of the human microbiome within the context of COVID-19 may offer valuable insights into disease
prevention, treatment strategies, and its potential applications in forensic science. Consequently, this paper aims
to provide an overarching review of microbiome alterations due to COVID-19 and the associated impact on
forensic applications, bridging the gap between the altered microbiome of COVID-19 patients and the challenges
forensic investigations may encounter when analyzing this microbiome as a forensic biomarker.
The relevance of postmortem microbiological examinations has been controversial for decades, but the boom in advanced sequencing techniques over the last decade is increasingly demonstrating their usefulness, namely for the estimation of the postmortem interval. This comprehensive review aims to present the current knowledge about the human postmortem microbiome (the necrobiome), highlighting the main factors influencing this complex process and discussing the principal applications in the field of forensic sciences. Several limitations still hindering the implementation of forensic microbiology, such as small-scale studies, the lack of a universal/harmonized workflow for DNA extraction and sequencing technology, variability in the human microbiome, and limited access to human cadavers, are discussed. Future research in the field should focus on identifying stable biomarkers within the dominant Bacillota and Pseudomonadota phyla, which are prevalent during postmortem periods and for which standardization, method consolidation, and establishment of a forensic microbial bank are crucial for consistency and comparability. Given the complexity of identifying unique postmortem microbial signatures for robust databases, a promising future approach may involve deepening our understanding of specific bacterial species/strains that can serve as reliable postmortem interval indicators during the process of body decomposition. Microorganisms might have the potential to complement routine forensic tests in judicial processes, requiring robust investigations and machine-learning models to bridge knowledge gaps and adhere to Locard’s principle of trace evidence.
Estimating the post-mortem time interval (PMI) is a crucial component of the forensic investigation and is extremelydifficult for medico-legal experts to do. After death, the succession of microbes in various parts of the human bodyhas enormous potential for predicting PMI. The human body is home to trillions of commensal microorganisms.These microbes behave in a different way when biological processes stop, which coincides with the death of anindividual and the invasion of deteriorating microbes from the environment. Due to cell autolysis, which draws avariety of invasive macro- and microorganisms, human cadavers become a rich source of nutrients. The successionof microorganisms differs significantly at various stages of degradation, which can be investigated for precisePMI estimation. Necrobiome analysis has drawn a lot of attention in PMI estimation due to the developmentof microbial genomics technology and decrease in the price of DNA sequencing. The review article provides asummary of the various microorganism sources, their successional pattern, and analytical methods that can beused in the field of microbial forensics.
Background
Microorganisms distribute and proliferate both inside and outside the body, which are the main mediators of decomposition after death. However, limited information is available on the postmortem microbiota changes of extraintestinal body sites in the early decomposition stage of mammalian corpses.
Results
This study investigated microbial composition variations among different organs and the relationship between microbial communities and time since death over 1 day of decomposition in male C57BL/6 J mice by 16S rRNA sequencing. During 1 day of decomposition, Agrobacterium, Prevotella, Bacillus, and Turicibacter were regarded as time-relevant genera in internal organs at different timepoints. Pathways associated with lipid, amino acid, carbohydrate and terpenoid and polyketide metabolism were significantly enriched at 8 h than that at 0.5 or 4 h. The microbiome compositions and postmortem metabolic pathways differed by time since death, and more importantly, these alterations were organ specific.
Conclusion
The dominant microbes differed by organ, while they tended toward similarity as decomposition progressed. The observed thanatomicrobiome variation by body site provides new knowledge into decomposition ecology and forensic microbiology. Additionally, the microbes detected at 0.5 h in internal organs may inform a new direction for organ transplantation.
As prophesized in early Tibetan medical works, the emergence of a pathogen such as SARS-CoV-2 that could inflict such a virulent infectious disease such as COVID-19 provided conditions for an expected yet alarming new phenomenon to threaten the health of inhabitants on the Tibetan Plateau. As SARS-CoV-2 spread into a global pandemic, Tibetan physicians worldwide engaged in symposiums, conferences, and clinical exchanges to situate the virus and its disease within Tibetan medical nosology. They sought to reconcile prophesies of global impact and develop critical treatment protocols for their communities. This article presents this particular perspective on COVID-19 as discussed among Tibetan medical colleagues in early April 2020, with follow-up discussions a year later. It introduces the disease’s nosology as a specific type of virulent infection (gnyan rims), and describes the etiology, diagnosis, and treatment as explicated in the Tibetan classic Four Medical Treatises and related commentaries. As Tibetan physicians gain attention for their treatments of mild- and medium-severity COVID-19 cases, understanding the Tibetan medical paradigm for the condition highlights distinctions of therapeutic and investigative relevance compared to biomedical and other traditional Asian medical approaches.
Nowadays, the diagnostic value of postmortem microbiological investigations is still a debated topic, but postmortem microbiology (PMM) remains a discipline with great forensic potential. To evaluate the usefulness and diagnostic-forensic value of postmortem microbiological cultures, it has been conducted a study on cadaveric material sampled during autopsy aiming to identify the correct cause of death. The study analyzed 45 cadavers subjected to judicial autopsy, divided into two groups based on the presence or absence of external or internal macroscopic autopsy signs suggesting infectious pathology. In the same cases, both the microbiological and conventional histological investigations have been simultaneously carried out. From the investigations, mono-bacterial, mono-fungal, mixed and negative cultures were observed. In mono-species microbiological growth, the histological epicrisis confirmed an infectious cause of death due to the presence of signs of acute infection with an aggressive infectious agent. In cases where growth was mixed, it was possible to distinguish between simple postmortal contamination and perimortem colonization. Finally, in some cases where the microbiology was negative, this has been essential in highlighting signs of a vital reaction to viral or parasitic infection. The joint and integrated evaluation of the laboratory results made it possible to correctly understand even those peculiar situations in which the PMM results alone would not have been significant. These methods, when combined, constitute an optimal forensic approach for the identification of the real cause of death and thus reduce the number of unsolved cases.
Introduction:
The human post-mortem microbiome (HPM) plays a major role in the decomposition process. Successional changes in post-mortem bacterial communities have been recently demonstrated using high throughput metagenomic sequencing techniques, showing great potential as a post-mortem interval (PMI) predictor. The aim of this study is to verify the application of the mass spectrometry technique, better known as MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), as a cheap and quick method for microbe taxonomic identification and for studying the PM microbiome.
Methods:
The study was carried out on 18 human bodies, ranging from 4 months to 82 years old and with a PMI range from 24 h up to 15 days. The storage time interval in the coolers was included in the final PMI estimates. Using the PMI, the sample study was divided into three main groups: seven cases with a PMI < 72 h; six cases with a PMI of 72-168 h and five cases with a PMI > 168 h. For each body, microbiological swabs were sampled from five external anatomical sites (eyes, ears, nose, mouth, and rectum) and four internal organs (brain, spleen, liver, and heart).
Results:
The HPM became increasingly different from the starting communities over time in the internal organs as well as at skin sites; the HPM microbiome was mostly dominated by Firmicutes and Proteobacteria phyla; and a PM microbial turnover existed during decomposition, evolving with the PMI.
Conclusions:
MALDI-TOF is a promising method for PMI estimation, given its sample handling, good reproducibility, and high speed and throughput. Although several intrinsic and extrinsic factors can affect the structure of the HPM, MALDI-TOF can detect the overall microbial community turnover of most prevalent phyla during decomposition. Limitations are mainly related to its sensitivity due to the culture-dependent method and bias in the identification of new isolates.
Pathologists have been concerned about the validity of bacterial cultures from lung tissue for the postmortem identification of pneumonia-associated pathogens. Therefore, we studied whether pathogenic bacteria that cause pneumonia could be identified via the metagenomic analysis of lung tissue sampled at autopsy from 11 pneumonia cases and nine non-pneumonia cases. We demonstrated that metagenomic analysis of the postmortem lung microbiota could identify a bacterial genus as a pneumonia pathogen when the genus was predominant and an established pathogen. However, it is important to diagnose Enterobacteriaceae or anaerobic bacteria as pneumonia pathogens, because their abundance in the lung microbiota of non-pneumonia cases supports postmortem translocation and replacement. Moreover, we confirmed that postmortem bacterial culture from lung tissue could be misleading for the identification of the pneumonia pathogen based on postmortem anaerobic changes and artificial culture conditions. To our knowledge, this study is the first to demonstrate the conditions under which metagenomic analysis of postmortem lung tissue can identify bacterial genera that cause pneumonia.
Through experimental reproduction of the decomposition process using the cadaver of rabbit Oryctolagus sp., the dynamics of cadaver microflora was studied; the dominant bacterial taxa were isolated in pure culture and identified; their ecological and trophic profiles and biodiversity were described based on the values of Simpson and Menchinic ecological indices. The dependence of cadaver rotting and skeletonization rate on the taxonomic profile of microorganisms, decomposition period, and abiotic environmental factors (temperature, acidity, soil moisture) was demonstrated. The data obtained contribute to justifying the use of microbiological methods in forensic practice. To establish objective causality patterns of microbial transformation of organic matter in nature, further targeted study of ecological patterns of cadaveric microflora is necessary.
Gradual changes in microbial communities in a human body after death can be used to determine postmortem interval (PMI). In this study, gut microflora samples were collected from the vermiform appendix and the transverse colon of human cadavers with PMIs between 5 and 192 h. The results revealed that the appendix might be an excellent intestinal sampling site and the appendix flora had an inferred succession rule during human body decomposition. Firmicutes, Bacteroidetes, and their respective subclasses showed a predictable succession rule in relative abundance over time. A Random Forest regression model was developed to correlate human gut microbiota with PMI. We believe that our findings have increased the knowledge of the composition and abundance of the gut microbiota in human corpses, and suggest that the use of the human appendix microbial succession may be a potential method for forensic estimation of the time of death.
Microbiological studies show that there is a possibility of PMI estimation in reference to presence of typical bacteria and fungi on cadaver or in soil beneath. Microbiome after death (thanatomicrobiome) changes and depends on time since death, temperature, seasons and environment-if human remains are covered, buried, placed in ice or left on the surface. To enlarge current knowledge, some of studies are conducted on animal models with further comparison thanatomicrobiome of different animals-pig, rats-to human cadaver thanatomicrobiome. This study collects different branches of thanatomicrobiome studies as a review to summarize current knowledge.
1. Introduction. 2. Living host microbiome and mycobiome. 3. Diseases-related differences. 4. Thanatomicrobiome – human cadavers studies. 5. Fungi presence – thanatomycobiome. 6. Thanatomicrobiome of frozen cadavers. 7. Soil microbial communities changes. 8. Seasons related microbial changes. 9. Thanatomicrobiome and entomology correlation. 10. Conclusions
Since the inception of evidence-based scientific concepts in medicine in the 19th century, the utility of postmortem microbiologic
examinations has been a topic of controversy. For every study describing a lack of correlation between antemortem clinical
and laboratory findings and postmortem culture results, there is equal evidence from other studies that indicates at least
some limited utility in select cases. While the contributions of autopsies and postmortem microbiologic examinations in the
discovery of novel infectious microorganisms are generally appreciated by the medical and scientific societies, the problems
of implementing routine procedures in daily autopsy practice clearly relate to the lack of consensus on their broader utility
as well as to a lack of regulatory guidelines. This review provides an overview of the literature-based evidence regarding
the utility of postmortem microbiologic examinations together with some practical aspects and guidelines for those confronted
with the issue of whether to allow or discourage the use of bacteriologic cultures obtained during autopsies.
Human decomposition is a mosaic system with an intimate association between biotic and abiotic factors. Despite the integral role of bacteria in the decomposition process, few studies have catalogued bacterial biodiversity for terrestrial scenarios. To explore the microbiome of decomposition, two cadavers were placed at the Southeast Texas Applied Forensic Science facility and allowed to decompose under natural conditions. The bloat stage of decomposition, a stage easily identified in taphonomy and readily attributed to microbial physiology, was targeted. Each cadaver was sampled at two time points, at the onset and end of the bloat stage, from various body sites including internal locations. Bacterial samples were analyzed by pyrosequencing of the 16S rRNA gene. Our data show a shift from aerobic bacteria to anaerobic bacteria in all body sites sampled and demonstrate variation in community structure between bodies, between sample sites within a body, and between initial and end points of the bloat stage within a sample site. These data are best not viewed as points of comparison but rather additive data sets. While some species recovered are the same as those observed in culture-based studies, many are novel. Our results are preliminary and add to a larger emerging data set; a more comprehensive study is needed to further dissect the role of bacteria in human 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.
The forensic potential of microorganisms is becoming increasingly apparent as a consequence of advances in molecular sciences and genomics. This review discusses instances in which microbes, and in particular bacteria, can impact upon forensic investigations. There is increasing evidence that humans have an extremely diverse ‘microbiome’ that may prove useful in determining ethnicity, country of origin, and even personal identity. The human microbiome differs between regions of the body and may prove useful for determining the nature of stains such as those caused by saliva and vaginal fluid: it may even be possible to link the stains to the person responsible for them. Similarly, the composition of the microbiome present in a soil sample may prove a useful indicator of geographic origin or as a means of linking people, animals, or objects together or to a specific location. Microorganisms are important in the decay process and also influence the presence and concentration of alcohol, drugs, and other chemicals of forensic relevance. There is also a possibility that the entry of microorganisms into the body during the agonal period may prove useful for the diagnosis of drowning. The transmission of infectious diseases, and in particular sexually-transmitted diseases, can provide evidence linking a victim and a suspect. Microorganisms that cause fatal infections are not always identified at the time of death and may lead to the death being considered ‘suspicious’. If a fatal infection can be linked to a hospital or medical procedure it can lead to prosecutions and therefore it is important to determine when and where an infection was acquired. Similarly, naturally acquired infections need to be distinguished from those that result from malicious transmission. Microorganisms can therefore provide evidence in many different forensic scenarios but most of the work is still at the experimental stage and there are therefore many opportunities for further research.
Lactic Acid Bacteria (LAB) are ancient organisms that cannot biosynthesize functional cytochromes, and cannot get ATP from respiration. Besides sugar fermentation, they evolved electrogenic decarboxylations and ATP-forming deiminations. The right balance between sugar fermentation and decarboxylation/deimination ensures buffered environments thus enabling LAB to survive in human gastric trait and colonize gut. A complex molecular cross-talk between LAB and host exists. LAB moonlight proteins are made in response to gut stimuli and promote bacterial adhesion to mucosa and stimulate immune cells. Similarly, when LAB are present, human enterocytes activate specific gene expression of specific genes only. Furthermore, LAB antagonistic relationships with other microorganisms constitute the basis for their anti-infective role. Histamine and tyramine are LAB bioactive catabolites that act on the CNS, causing hypertension and allergies. Nevertheless, some LAB biosynthesize both gamma-amino-butyrate (GABA), that has relaxing effect on gut smooth muscles, and beta-phenylethylamine, that controls satiety and mood. Since LAB have reduced amino acid biosynthetic abilities, they developed a sophisticated proteolytic system, that is also involved in antihypertensive and opiod peptide generation from milk proteins. Short-chain fatty acids are glycolytic and phosphoketolase end-products, regulating epithelial cell proliferation and differentiation. Nevertheless, they constitute a supplementary energy source for the host, causing weight gain. Human metabolism can also be affected by anabolic LAB products such as conjugated linoleic acids (CLA). Some CLA isomers reduce cancer cell viability and ameliorate insulin resistance, while others lower the HDL/LDL ratio and modify eicosanoid production, with detrimental health effects. A further appreciated LAB feature is the ability to fix selenium into seleno-cysteine. Thus, opening interesting perspectives for their utilization as antioxidant nutraceutical vectors.
Early scientific research into "putrefaction" by eighteenth century physicians was driven by a need to understand and treat living patients who were suffering from "putrid diseases" (presumably conditions such as treponemal disease, non-specific osteomyelitus, bacterial skin infections, abscesses, and the like, which could result in the formation of necrotic tissue, but which today can be treated by modern medicine).1,2 But these works clearly recognized and tried to seek explanation to some of the fundamental microbially induced changes in the human body, in particular, to soft tissue that occur during different stages in the decomposition process and which result in pH change, and the evolution of volatile compounds. As such, these works are an early precursor to the discipline that today we know as "taphonomy". This term, originally coined by the Russian palaeontologist Ivan Efremov to describe the "transformations from the biosphere to the lithosphere"3 in explaining the formation of fossils, today has much broader meaning. The term has been widely adopted in archaeology and forensic science and is concerned with the decomposition of the body and associated death scene materials. As such, the disciplines of archaeological taphonomy/diagenesis4-7 and forensic taphonomy 8-11 cover the location of buried or disturbed human remains 12 and time since death/burial estimation, and explain the survival/differential decomposition of physical remains and macromolecules such as proteins, lipids, and DNA. © Springer Science+Business Media, LLC 2009. All rights reserved.
Volatile emission during vertebrate decay is a complex process that is understood incompletely. It depends on many factors. The main factor is the metabolism of the microbial species present inside and on the vertebrate. In this review, we combine the results from studies on volatile organic compounds (VOCs) detected during this decay process and those on the biochemical formation of VOCs in order to improve our understanding of the decay process. Micro-organisms are the main producers of VOCs, which are by- or end-products of microbial metabolism. Many microbes are already present inside and on a vertebrate, and these can initiate microbial decay. In addition, micro-organisms from the environment colonize the cadaver. The composition of microbial communities is complex, and communities of different species interact with each other in succession. In comparison to the complexity of the decay process, the resulting volatile pattern does show some consistency. Therefore, the possibility of an existence of a time-dependent core volatile pattern, which could be used for applications in areas such as forensics or food science, is discussed. Possible microbial interactions that might alter the process of decay are highlighted.
A colony PCR technique was applied for both genomic and chloroplast DNA in the green microalgae Chlorella. Of five different lysis buffers, Chelex-100 was superior for DNA extraction, PCR and DNA storage. It also was insensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically-engineered transformants as well as bioprospecting of natural microalgal isolates. Besides multiple Chlorella species, we also demonstrate the efficacy of Chelex-100 for colony PCR with a number of other microalgal strains, including Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis sp., Coccomyxa sp., and Thalassiosira pseudonana.
Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80 degrees C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method.
Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume
efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify
this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA
and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter
polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior,
in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The
simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community.
The Human Microbiome Project (HMP), funded as an initiative of the NIH Roadmap for Biomedical Research (http://nihroadmap.nih.gov), is a multi-component community resource. The goals of the HMP are: (1) to take advantage of new, high-throughput technologies to characterize the human microbiome more fully by studying samples from multiple body sites from each of at least 250 "normal" volunteers; (2) to determine whether there are associations between changes in the microbiome and health/disease by studying several different medical conditions; and (3) to provide both a standardized data resource and new technological approaches to enable such studies to be undertaken broadly in the scientific community. The ethical, legal, and social implications of such research are being systematically studied as well. The ultimate objective of the HMP is to demonstrate that there are opportunities to improve human health through monitoring or manipulation of the human microbiome. The history and implementation of this new program are described here.
The human distal gut harbours a vast ensemble of microbes (the microbiota) that provide important metabolic capabilities, including the ability to extract energy from otherwise indigestible dietary polysaccharides. Studies of a few unrelated, healthy adults have revealed substantial diversity in their gut communities, as measured by sequencing 16S rRNA genes, yet how this diversity relates to function and to the rest of the genes in the collective genomes of the microbiota (the gut microbiome) remains obscure. Studies of lean and obese mice suggest that the gut microbiota affects energy balance by influencing the efficiency of calorie harvest from the diet, and how this harvested energy is used and stored. Here we characterize the faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity, and their mothers, to address how host genotype, environmental exposure and host adiposity influence the gut microbiome. Analysis of 154 individuals yielded 9,920 near full-length and 1,937,461 partial bacterial 16S rRNA sequences, plus 2.14 gigabases from their microbiomes. The results reveal that the human gut microbiome is shared among family members, but that each person's gut microbial community varies in the specific bacterial lineages present, with a comparable degree of co-variation between adult monozygotic and dizygotic twin pairs. However, there was a wide array of shared microbial genes among sampled individuals, comprising an extensive, identifiable 'core microbiome' at the gene, rather than at the organismal lineage, level. Obesity is associated with phylum-level changes in the microbiota, reduced bacterial diversity and altered representation of bacterial genes and metabolic pathways. These results demonstrate that a diversity of organismal assemblages can nonetheless yield a core microbiome at a functional level, and that deviations from this core are associated with different physiological states (obese compared with lean).
Random community genomes (metagenomes) are now commonly used to study microbes in different environments. Over the past few years, the major challenge associated with metagenomics shifted from generating to analyzing sequences. High-throughput, low-cost next-generation sequencing has provided access to metagenomics to a wide range of researchers.
A high-throughput pipeline has been constructed to provide high-performance computing to all researchers interested in using metagenomics. The pipeline produces automated functional assignments of sequences in the metagenome by comparing both protein and nucleotide databases. Phylogenetic and functional summaries of the metagenomes are generated, and tools for comparative metagenomics are incorporated into the standard views. User access is controlled to ensure data privacy, but the collaborative environment underpinning the service provides a framework for sharing datasets between multiple users. In the metagenomics RAST, all users retain full control of their data, and everything is available for download in a variety of formats.
The open-source metagenomics RAST service provides a new paradigm for the annotation and analysis of metagenomes. With built-in support for multiple data sources and a back end that houses abstract data types, the metagenomics RAST is stable, extensible, and freely available to all researchers. This service has removed one of the primary bottlenecks in metagenome sequence analysis - the availability of high-performance computing for annotating the data. http://metagenomics.nmpdr.org.
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B. subtilis subsp. niger) endospores or Fusarium moniliforme conidia. The hot-detergent-bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells or B. globigii spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of P. putida cells and B. globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.
For a long time necrosis was considered as an alternative to programmed cell death, apoptosis. Indeed, necrosis has distinct morphological features and it is accompanied by rapid permeabilization of plasma membrane. However, recent data indicate that, in contrast to necrosis caused by very extreme conditions, there are many examples when this form of cell death may be a normal physiological and regulated (programmed) event. Various stimuli (e.g., cytokines, ischemia, heat, irradiation, pathogens) can cause both apoptosis and necrosis in the same cell population. Furthermore, signaling pathways, such as death receptors, kinase cascades, and mitochondria, participate in both processes, and by modulating these pathways, it is possible to switch between apoptosis and necrosis. Moreover, antiapoptotic mechanisms (e.g., Bcl-2/Bcl-x proteins, heat shock proteins) are equally effective in protection against apoptosis and necrosis. Therefore, necrosis, along with apoptosis, appears to be a specific form of execution phase of programmed cell death, and there are several examples of necrosis during embryogenesis, a normal tissue renewal, and immune response. However, the consequences of necrotic and apoptotic cell death for a whole organism are quite different. In the case of necrosis, cytosolic constituents that spill into extracellular space through damaged plasma membrane may provoke inflammatory response; during apoptosis these products are safely isolated by membranes and then are consumed by macrophages. The inflammatory response caused by necrosis, however, may have obvious adaptive significance (i.e., emergence of a strong immune response) under some pathological conditions (such as cancer and infection). On the other hand, disturbance of a fine balance between necrosis and apoptosis may be a key element in development of some diseases.
To assess the value of postmortem bacteriology in necropsy practice, with specific emphasis on bacterial invasion of blood and cerebrospinal fluid (CSF).
A review of published articles on postmortem bacteriology. Studies were selected to cover the full range of necropsy practice including adults, the perinatal period, and infancy. The review covers over 5000 necropsies, mainly in adults, but including 1108 perinatal cases and 468 cases of sudden unexpected death in infancy. Data are available on 4992 blood cultures, 1168 specimens of CSF, and 743 cultures of spleen.
Studies in which careful precautions have been taken to reduce contamination show that approximately two thirds of blood cultures are negative, two in nine yield a single isolate, and one in nine have a mixed growth. The postmortem interval has only a small effect on the isolation rate. A pure growth of a known pathogen has a more than 50% likelihood of being found in association with genuine infection in adults and in the perinatal period.
The main postmortem artefact is contamination, but this can be considerably reduced by careful technique. Agonal spread is less common than is often assumed. Postmortem translocation is not a problem if the body is appropriately stored. A pure growth of a pathogen in blood or CSF should be regarded as a possible contributing factor to death at all ages.
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
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.
Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims.
The process of decomposition of bodies in the marine environment is poorly understood and almost nothing is currently known about the microorganisms involved. This study aimed to investigate the microbes involved in decomposition in the sea and to evaluate the potential use of marine bacterial succession for postmortem submersion interval (PMSI) estimation, for which there is currently no reliable method. Partial pig remains were completely submerged during autumn and winter and were regularly sampled to document marine bacterial colonisation and the changes in community composition over time. Five stages of decomposition were recognised, some of which exhibited characters specific for partial carrion. Marine bacteria rapidly colonised the submerged remains in a successional manner. Seasonal differences were observed for the rate of decomposition and also for several groups of colonising bacteria. Marine bacteria specific for particular PMSIs were identified. This study provides an insight into the involvement of saprophytic marine bacteria in the decomposition of mammalian remains in the sea and is the first to explore the use of marine bacterial colonisation and succession as a novel tool for PMSI estimation. We propose that with further study, marine bacterial succession will prove useful for determination of the length of time a body may have been immersed in a marine environment.
The postomortem invasion of muscle and other tissues by bacteria from the intestinal tract was studied with the use of radioactive tracers. The injection of 14C-labeled bacteria or spores into the intestines of guinea pig carcasses within 24 h of death resulted in the rapid spread of 14C throughout carcasses. When live bacteria were injected along with the labeled cells, it was not possible to isolate viable organisms from the body tissues if the living animal had been exposed to the bacteria. It appears that animals are immune to their normal intestinal flora and that this immunity persists after death; thus passage of these bacteria into the lymphatic system does not necessarily result in the presence of live bacteria in carcass tissues. It therefore seems that a delay of up to 24 h before evisceration would not lead to deep tissue contamination of the carcass by organisms usually present in the intestines. Further evidence for this hypothesis was obtained by showing that muscle and lymph nodes from uneviscerated lamb carcasses hung for 24 h at 20 C remained sterile.
Studies of cell injury and death at the molecular and cell-biological level will help to provide methods for the reconstruction of the course of events between the occurrence of primary pathology and the time of investigation. The general role of component turnover, energy metabolism and lysosomes in cellular injury is briefly described.
This study was conducted to characterize the chemistry associated with the decomposition of human remains with the objective of identifying time-dependent biomarkers of decomposition. The purpose of this work was to develop an accurate and precise method for measuring the postmortem interval (PMI) of human remains. Eighteen subjects were placed within a decay research facility throughout a four-year time period and allowed to decompose naturally. Field autopsies were performed and tissue samples were regularly collected until the tissues decomposed to the point where they were no longer recognizable (encompassing a cumulative degree hour (CDH) range of approximately 1000 (approximately 3 weeks)). Analysis of the biomarkers (amino acids, neurotransmitters, and decompositional by-products) in various organs (liver, kidney, heart, brain, muscle) revealed distinct patterns useful for determining the PMI when based on CDHs. Proper use of the methods described herein allow for PMIs so accurate that the estimate is limited by the ability to obtain correct temperature data at a crime scene rather than sample variability.
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Bacteremias in the agonal period
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A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system
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