Microbial succession in human rib skeletal remains and fly-human microbial transfer during decomposition

2022 Spring.Includes bibliographical references.Human decomposition is a dynamic process partially driven by the actions of microbes. It can be defined by the fresh, early decomposition, advanced decomposition, and skeletonization stages. The microbial communities that facilitate decomposition change in a predictable, clock-like manner, which can be used as a forensic tool for estimating postmortem interval. Chapter 1 introduces this concept by describing the stages of decomposition in detail and how high-throughput sequencing methods can be used with microbes to develop models for predicting postmortem interval. Chapter 1 also describes which sample types are most useful for predicting postmortem interval based on the stage of decomposition, the knowledge gaps in the field, and the steps necessary for adoption of this tool into the justice system. During fresh and early decomposition, microbial succession of the skin and soil sample types are most predictive of postmortem interval. However, after approximately the first three weeks of decomposition, the changes in the microbial communities that are used for predictions begin to slow down and the skin and soil sample types become less useful for estimating postmortem interval. Chapter 2 of this dissertation shows that microbial succession of the bone microbial decomposer communities can be used for estimating postmortem interval during the advanced and skeletonization stages of decomposition. First, the bone microbial decomposer community was characterized using 16S ribosomal RNA sequencing from six human donor subjects placed in the spring and summer seasons at the Southeast Texas Applied Forensic Science Facility. A core bone decomposer microbiome dominated by taxa within phylum Proteobacteria was discovered, as well as significant overall differences in the bone microbial community between the spring and summer seasons. These microbial community data were used to develop random forest models that predicted postmortem interval within +/- 34 days over a 1–9-month time frame of decomposition. To gain a better understanding of where the microbes in the decomposed bone were coming from, as healthy, living bone is typically sterile, SourceTracker2 was used with paired skin and soil samples taken from the same decedents. Results showed that the bone microbial decomposer community is likely sourced from the surrounding environment, particularly the skin and soil communities that occur during the advanced stage of decomposition. Chapter 3 of this dissertation focuses on the influence of the blow fly (Calliphoridae) microbiome on human cadaver microbial community assembly. In early decomposition, volatiles attract blow flies to the cadaver, which serves as a source of nutrients and a safe place to lay eggs. It is likely that during this interaction between hosts, there is a mechanical transfer of microbes that subsequently alters each of their microbial communities. While studies have shown that blow flies have their own microbiome, they were not conducted in a decomposition environment. First, Chapter 3 shows the characterization of the blow fly microbiome by organ and season in a terrestrial, human decomposition environment. This was performed by placing ten cadavers across the winter, spring, and summer seasons at the Southeast Texas Applied Forensic Science Facility, collecting the first wave of colonizing flies for each cadaver, and sequencing the 16S ribosomal RNA gene of the labellum (mouth parts), tarsi (leg parts), and oocytes. Results showed that the previously defined universal fly microbiome persists even in a decomposition environment, with notable differences still present between organs and seasons. Additionally, results from using the tool SourceTracker2 showed that the labellum and tarsi act as substantial bacterial sources of the human decomposer bacterial community, and this source contribution varies by season. In summary, this dissertation provides the first quantitative estimate of postmortem interval of terrestrially decomposed human skeletal remains using microbial abundance information. This is a significant contribution to the criminal justice system; anthropologists typically use visual evidence to provide postmortem interval estimates of skeletal remains with errors ranging from months to years, whereas our approach provides estimates with errors of approximately one month. Furthermore, this dissertation shows evidence that there is a mechanical transfer of microbes between blow flies and human cadavers during the early stage of decomposition, which provides ecological insight into human cadaver microbial community assembly

A Pilot Study of Microbial Succession in Human Rib Skeletal Remains during Terrestrial Decomposition.

The bones of decomposing vertebrates are colonized by a succession of diverse microbial communities. If this succession is similar across individuals, microbes may provide clues about the postmortem interval (PMI) during forensic investigations in which human skeletal remains are discovered. Here, we characterize the human bone microbial decomposer community to determine whether microbial succession is a marker for PMI. Six human donor subjects were placed outdoors to decompose on the soil surface at the Southeast Texas Applied Forensic Science facility. To also assess the effect of seasons, three decedents were placed each in the spring and summer. Once ribs were exposed through natural decomposition, a rib was collected from each body for eight time points at 3 weeks apart. We discovered a core bone decomposer microbiome dominated by taxa in the phylum Proteobacteria and evidence that these bone-invading microbes are likely sourced from the surrounding decomposition environment, including skin of the cadaver and soils. Additionally, we found significant overall differences in bone microbial community composition between seasons. Finally, we used the microbial community data to develop random forest models that predict PMI with an accuracy of approximately ±34 days over a 1- to 9-month time frame of decomposition. Typically, anthropologists provide PMI estimates based on qualitative information, giving PMI errors ranging from several months to years. Previous work has focused on only the characterization of the bone microbiome decomposer community, and this is the first known data-driven, quantitative PMI estimate of terrestrially decomposed human skeletal remains using microbial abundance information. IMPORTANCE 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. However, when remains are discovered after months or years, often the only evidence are skeletal remains. To determine if microbial succession in bone would be useful for estimating time since death after several months, human subjects were placed to decompose in the spring and summer seasons. Ribs were collected after 1 to 9 months of decomposition, and the bone microbial communities were characterized. Analysis revealed a core bone decomposer microbial community with some differences in microbial assembly occurring between seasons. These data provided time since death estimates of approximately ±34 days over 9 months. This may provide forensic investigators with a tool for estimating time since death of skeletal remains, for which there are few current methods

A conserved interdomain microbial network underpins cadaver decomposition despite environmental variables.

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

Experiences and lessons learned from two virtual, hands-on microbiome bioinformatics workshops.

In October of 2020, in response to the Coronavirus Disease 2019 (COVID-19) pandemic, our team hosted our first fully online workshop teaching the QIIME 2 microbiome bioinformatics platform. We had 75 enrolled participants who joined from at least 25 different countries on 6 continents, and we had 22 instructors on 4 continents. In the 5-day workshop, participants worked hands-on with a cloud-based shared compute cluster that we deployed for this course. The event was well received, and participants provided feedback and suggestions in a postworkshop questionnaire. In January of 2021, we followed this workshop with a second fully online workshop, incorporating lessons from the first. Here, we present details on the technology and protocols that we used to run these workshops, focusing on the first workshop and then introducing changes made for the second workshop. We discuss what worked well, what didn't work well, and what we plan to do differently in future workshops

Experiences and lessons learned from two virtual, hands-on microbiome bioinformatics workshops

In October of 2020, in response to the Coronavirus Disease 2019 (COVID-19) pandemic, our team hosted our first fully online workshop teaching the QIIME 2 microbiome bioinformatics platform. We had 75 enrolled participants who joined from at least 25 different countries on 6 continents, and we had 22 instructors on 4 continents. In the 5-day workshop, participants worked hands-on with a cloud-based shared compute cluster that we deployed for this course. The event was well received, and participants provided feedback and suggestions in a postworkshop questionnaire. In January of 2021, we followed this workshop with a second fully online workshop, incorporating lessons from the first. Here, we present details on the technology and protocols that we used to run these workshops, focusing on the first workshop and then introducing changes made for the second workshop. We discuss what worked well, what didn’t work well, and what we plan to do differently in future workshops.ISSN:1553-734XISSN:1553-735