Microbial degradation of organic matter is a fundamental Earth process, yet a mechanistic understanding of microbial metabolisms and successional ecology involved in decomposition remains poorly understood. Here, we announce the recovery of 277 cadaver-associated soil metagenome-assembled genomes to enhance our understanding of vertebrate decomposition microbial processes.

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

During decomposition, flies interact with the remains to lay eggs and acquire nutrients, and in the process, they bring their microbes with them. While it is known that flies have their own unique core microbiome, it is not known if flies associated with human cadavers have a different core microbiome. Differences in the fly microbiome may influence the types of microbes transmitted from the flies to the cadaver, therefore potentially affecting assembly of the human decomposer microbiome. The first purpose of this study was to characterize the microbiome of flies associated with human cadavers by fly organ and season. This is because fly interactions with cadavers vary by season, and because it is likely that external fly organs [i.e., the labellum and tarsi] make more direct contact and are likely involved in increased mechanical transmission with the cadaver than internal organs such as the oocyte. The second purpose of this study was to determine if the fly microbes contribute to the human decomposer microbiome. To accomplish these aims, 10 human cadavers were placed outdoors across three seasons and allowed to decompose. A total of 40 flies that landed on the cadaver were collected and dissected by the labellum, tarsi, and oocyte. In addition to fly collections, samples from the cadavers were collected using a sterile swab at sites including the cheek of the face, inner cheek, bicep, torso, and anus. Overall, it was shown that flies associated with human cadavers have a similar microbiome to flies from previous studies that were not associated with human cadavers. However, there are differences in the microbiome between seasons and fly parts. We also show evidence that flies act as a microbial source to the human decomposer microbiome, which is important for understanding the ecological mechanisms of human cadaver microbial community assembly.

Microbes are known to facilitate vertebrate decomposition, and they can do so in a repeatable, predictable manner. The succession of microbes in the skin and associated soil can be used to predict time since death during the first few weeks of decomposition.

Estimating the postmortem interval (PMI) of human remains is important in criminal investigations. Microbes play an important role in the process of decomposition and can provide clues about the time elapsed since death. Host-associated and environmental microbial communities have been shown to undergo succession in a predictable, clock-like manner during decomposition. High-throughput DNA sequencing can be used to inexpensively and rapidly track these microbial community shifts, and machine learning techniques can use these data to develop predictive models. In this chapter, we discuss the development of a microbial clock for estimating PMI, as well as remaining knowledge gaps and hurdles to technology adoption.

Death investigations often include an effort to establish the postmortem interval (PMI) in cases in which the time of death is uncertain. The postmortem interval can lead to the identification of the deceased and the validation of witness statements and suspect alibis. Recent research has demonstrated that microbes provide an accurate clock that starts at death and relies on ecological change in the microbial communities that normally inhabit a body and its surrounding environment. Here, we explore how to build the most robust Random Forest regression models for prediction of PMI by testing models built on different sample types (gravesoil, skin of the torso, skin of the head), gene markers (16S ribosomal RNA (rRNA), 18S rRNA, internal transcribed spacer regions (ITS)), and taxonomic levels (sequence variants, species, genus, etc.). We also tested whether particular suites of indicator microbes were informative across different datasets. Generally, results indicate that the most accurate models for predicting PMI were built using gravesoil and skin data using the 16S rRNA genetic marker at the taxonomic level of phyla. Additionally, several phyla consistently contributed highly to model accuracy and may be candidate indicators of PMI.

Deep in the primordial forest of eastern Texas, we watch our team of undergraduate and graduate students taking swab samples from a pair of human cadavers that we placed at the Southeast Texas Applied Forensics Science (STAFS) facility of Sam Houston State University (SHSU) in Huntsville. Our highly choreographed sampling regime, which focuses on bacterial communities, includes swabbing seven body sites on each cadaver, collecting nearby soil samples, tabulating temperatures, and collecting cadaver-associated flies. We do this daily until the bodies begin to dry out, then sample every other day for another month, then once weekly for yet another month, then once per month for 6 months until only dry bones remain. We continue taking soil samples for approximately one year longer.

Vertebrate corpse decomposition provides an important stage in nutrient cycling in most terrestrial habitats, yet microbially mediated processes are poorly understood. Here we combine deep microbial community characterization, community-level metabolic reconstruction, and soil biogeochemical assessment to understand the principles governing microbial community assembly during decomposition of mouse and human corpses on different soil substrates. We find a suite of bacterial and fungal groups that contribute to nitrogen cycling and a reproducible network of decomposers that emerge on predictable time scales. Our results show that this decomposer community is derived primarily from bulk soil, but key decomposers are ubiquitous in low abundance. Soil type was not a dominant factor driving community development, and the process of decomposition is sufficiently reproducible to offer new opportunities for forensic investigations.

Human decomposition is a dynamic process that is influenced by both abiotic and biotic factors. Measuring these influences, in particular abiotic factors, on the decomposition process is often a challenge for scientists. Recently, researchers have turned to the use of advanced remote sensing technologies in forensic investigations. In this study, a new methodology is described that utilizes precise 3D images captured using terrestrial laser scanning (TLS) to calculate total solar irradiance on a cadaver in a partially forested environment. To test this new measurement approach under actual field conditions, three cadavers were placed in an outdoor environment to decompose. Laser scans were taken the day of placement and used to calculate the total solar irradiance at time points of 24 h, 1 week, and 1 month from placement. The results show that as time progresses, different cadavers at the field site and different areas of the same cadaver receive varying amounts of solar irradiance. The modeling based on these laser scans can be used to create predictive images of solar irradiance that may provide researchers with a new tool to help quantitatively assess the effect of solar irradiance on a cadaver ecosystem.

Decomposition is a dynamic ecological process dependent upon many factors such as environment, climate, and bacterial, insect, and vertebrate activity in addition to intrinsic properties inherent to individual cadavers. Although largely attributed to microbial metabolism, very little is known about the bacterial basis of human decomposition. To assess the change in bacterial community structure through time, bacterial samples were collected from several sites across two cadavers placed outdoors to decompose and analyzed through 454 pyrosequencing and analysis of variable regions 3–5 of the bacterial 16S ribosomal RNA (16S rRNA) gene. Each cadaver was characterized by a change in bacterial community structure for all sites sampled as time, and decomposition, progressed. Bacteria community structure is variable at placement and before purge for all body sites. At bloat and purge and until tissues began to dehydrate or were removed, bacteria associated with flies, such as Ignatzschineria and Wohlfahrtimonas, were common. After dehydration and skeletonization, bacteria associated with soil, such as Acinetobacter, were common at most body sites sampled. However, more cadavers sampled through multiple seasons are necessary to assess major trends in bacterial succession.

Decomposition can be a highly variable process with stages that are difficult to quantify. Using high accuracy terrestrial laser scanning a repeated three-dimensional (3D) documentation of volumetric changes of a human body during early decomposition is recorded. To determine temporal volumetric variations as well as 3D distribution of the changed locations in the body over time, this paper introduces the use of multiple degenerated cylinder models to provide a reasonable approximation of body parts against which 3D change can be measured and visualized. An iterative closest point algorithm is used for 3D registration, and a method for determining volumetric change is presented. Comparison of the laser scanning estimates of volumetric change shows good agreement with repeated in-situ measurements of abdomen and limb circumference that were taken diurnally. The 3D visualizations of volumetric changes demonstrate that bloat is a process with a beginning, middle, and end rather than a state of presence or absence. Additionally, the 3D visualizations show conclusively that cadaver bloat is not isolated to the abdominal cavity, but also occurs in the limbs. Detailed quantification of the bloat stage of decay has the potential to alter how the beginning and end of bloat are determined by researchers and can provide further insight into the effects of the ecosystem on decomposition.

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.

Salmonella enterica serovar Heidelberg (S. Heidelberg) can cause foodborne illness in humans following the consumption of contaminated meat and poultry products. Recent studies from our laboratory have demonstrated that certain S. Heidelberg isolated from food-animal sources harbor multiple transmissible plasmids with genes that encode antimicrobial resistance, virulence and a VirB4/D4 type-IV secretion system. This study examines the potential role of these transmissible plasmids in bacterial uptake and survival in intestinal epithelial cells and macrophages, and the molecular basis of host immune system modulation that may be associated with disease progression. A series of transconjugant and transformant strains were developed with different combinations of the plasmids to determine the roles of the individual and combinations of plasmids on virulence. Overall the Salmonella strains containing the VirB/D4 T4SS plasmids entered and survived in epithelial cells and macrophages to a greater degree than those without the plasmid, even though they carried other plasmid types. During entry in macrophages, the VirB/D4 T4SS encoding genes are up-regulated in a time-dependent fashion. When the potential mechanisms for increased virulence were examined using an antibacterial Response PCR Array, the strain containing the T4SS down regulated several host innate immune response genes which likely contributed to the increased uptake and survival within macrophages and epithelial cells.

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopachologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 mu g of GST-Iss, and 1/12 chickens immunized with 50 mu g of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.

Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc) A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

Writing is a learning activity, as well as a communication skill. Many instructors recognize the value of writing as a learning tool but struggle to develop effective writing assignments. Instructors are generally pressed for time during lecture due to the necessity to deliver content and, therefore, cannot dedicate time necessary to teach science writing skills effectively. Traditional term papers assigned to a class with varying writing skills may not accomplish the desired goal of teaching both technical writing skills and critical thinking skills. Students that are already struggling with content may be at a disadvantage in terms of conveying complex ideas. An answer to this problem is the microtheme paper which we employ in an Introductory Botany laboratory setting.