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1. Conceptualizing the Gut Thanatomicrobiota in Substance Abuse Disorders

   Javan, GT ; Finley, SJ ; Scott, L ; Visonà, S. ( February 2021 , Proceedings American Academy of Forensic Sciences)

   null (Ed.)

   In this century, drug abuse continues to be a national crisis. Since 1999, the number of opioid-induced overdoses has increased four-fold to more than 500,000 deaths. The microbiota gut-brain-axis is a bidirectional circuit that links the neural, endocrine, and immunological systems with gut microbial communities. Gut microbiota play significant roles in human mind and behavior, specifically pain perception, learning capacity and memory, mood, and emotion, and anxiolytic effects and temperament. Also, disruptions in the gut microbiome have been associated with substance use disorders. While much research still needs to be performed, elucidating the interplay of gut microbiota in substance abuse disorders may produce promising avenues for future forensic development. The goal of the current study was to determine gut microbiome composition in substance abuse disorder cases using transverse colon tissues of 21 overdose criminal cases versus 19 non-overdose-related cases. The hypothesis was that postmortem samples of the same origin will reveal similar taxonomic relationships. Using weighted UniFrac analysis, drug abuse was found to be a significant factor in determining microbiome similarity (F = 1.93; df = 1, 35; p < 0.048; R2 = 0.05) indicating that there are detectable differences in composition that are attributable to substance abuse. Using unweighted UniFrac, however, sex was instead found to be a significant predictor of microbiome similarity (F = 1.88; df = 1, 30; p = 0.028; R2 = 0.05). A heatmap was generated of the relative abundances of the 30 most prevalent bacteria per case and their associated substance profile. The results revealed that samples of the same origin cluster together, showing a high degree of similarity between samples and a low degree of similarity among samples of different origin. This examination of human transverse colon microflora in decomposing cadavers expands the emerging literature on postmortem microbial communities, which will ultimately contribute to advanced knowledge of putrefaction. 

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2. Effects of Extended Postmortem Interval on Microbial Communities in Organs of the Human Cadaver

   https://doi.org/https://doi.org/10.3389/fmicb.2020.569630  

   Lutz, H ; Vangelatos, A ; Gottel, N ; Osculati, A ; Visona, S ; Finley, SJ ; Gilbert, J ; Javan, GT. ( December 2020 , Frontiers in microbiology)

   null (Ed.)

   Human thanatomicrobiota studies have shown that microorganisms inhabit and proliferate externally and internally throughout the body and are the primary mediators of putrefaction after death. Yet little is known about the source and diversity of the thanatomicrobiome or the underlying factors leading to delayed decomposition exhibited by reproductive organs. The use of the V4 hypervariable region of bacterial 16S rRNA gene sequences for taxonomic classification (“barcoding”) and phylogenetic analyses of human postmortem microbiota has recently emerged as a possible tool in forensic microbiology. The goal of this study was to apply a 16S rRNA barcoding approach to investigate variation among different organs, as well as the extent to which microbial associations among different body organs in human cadavers can be used to predict forensically important determinations, such as cause and time of death. We assessed microbiota of organ tissues including brain, heart, liver, spleen, prostate, and uterus collected at autopsy from criminal casework of 40 Italian cadavers with times of death ranging from 24 to 432 h. Both the uterus and prostate had a significantly higher alpha diversity compared to other anatomical sites, and exhibited a significantly different microbial community composition from non-reproductive organs, which we found to be dominated by the bacterial orders MLE1-12, Saprospirales, and Burkholderiales. In contrast, reproductive organs were dominated by Clostridiales, Lactobacillales, and showed a marked decrease in relative abundance of MLE1-12. These results provide insight into the observation that the uterus and prostate are the last internal organs to decay during human decomposition. We conclude that distinct community profiles of reproductive versus non-reproductive organs may help guide the application of forensic microbiology tools to investigations of human cadavers. 

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3. Macrobdella decora : Old World Leech Gut Microbial Community Structure Conserved in a New World Leech

   https://doi.org/10.1128/AEM.02082-20  

   McClure, Emily Ann ; Nelson, Michael C. ; Lin, Amy ; Graf, Joerg ( April 2021 , Applied and Environmental Microbiology)

   Johnson, Karyn N. (Ed.)

   ABSTRACT Leeches are found in terrestrial, aquatic, and marine habitats on all continents. Sanguivorous leeches have been used in medicine for millennia. Modern scientific uses include studies of neurons, anticoagulants, and gut microbial symbioses. Hirudo verbana , the European medicinal leech, maintains a gut community dominated by two bacterial symbionts, Aeromonas veronii and Mucinivorans hirudinis , which sometimes account for as much as 97% of the total crop microbiota. The highly simplified gut anatomy and microbiome of H. verbana make it an excellent model organism for studying gut microbial dynamics. The North American medicinal leech, Macrobdella decora , is a hirudinid leech native to Canada and the northern United States. In this study, we show that M. decora symbiont communities are very similar to those in H. verbana. We performed an extensive study using field-caught M. decora and purchased H. verbana from two suppliers. Deep sequencing of the V4 region of the 16S rRNA gene allowed us to determine that the core microbiome of M. decora consists of Bacteroides , Aeromonas, Proteocatella , and Butyricicoccus. The analysis revealed that the compositions of the gut microbiomes of the two leech species were significantly different at all taxonomic levels. The R 2 value was highest at the genus and amplicon sequence variant (ASV) levels and much lower at the phylum, class, and order levels. The gut and bladder microbial communities were distinct. We propose that M. decora is an alternative to H. verbana for studies of wild-caught animals and provide evidence for the conservation of digestive-tract and bladder symbionts in annelid models. IMPORTANCE Building evidence implicates the gut microbiome in critical animal functions such as regulating digestion, nutrition, immune regulation, and development. Simplified, phylogenetically diverse models for hypothesis testing are necessary because of the difficulty of assigning causative relationships in complex gut microbiomes. Previous research used Hirudo verbana as a tractable animal model of digestive-tract symbioses. Our data show that Macrobdella decora may work just as well without the drawback of being an endangered organism and with the added advantage of easy access to field-caught specimens. The similarity of the microbial community structures of species from two different continents reveals the highly conserved nature of the microbial symbionts in sanguivorous leeches. 

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4. Postmortem succession of gut microbial communities in deceased human subjects

   https://doi.org/10.7717/peerj.3437  

   DeBruyn, Jennifer M. ; Hauther, Kathleen A. ( June 2017 , PeerJ)

   The human microbiome has demonstrated an importance for the health and functioning in living individuals. However, the fate of the microbiome after death is less understood. In addition to a better understanding of microbe-mediated decomposition processes, postmortem succession of human-associated microbial communities has been suggested as a possible forensic tool for estimating time since death, or postmortem interval (PMI). The objective of our study was to document postmortem changes in human gut bacterial communities. Gut microflora were repeatedly sampled from the caeca of cadavers as they decayed under natural environmental conditions. 16S rRNA gene amplicon sequencing revealed that over time, bacterial richness significantly increased (r_s = 0.449) while diversity decreased (r_s =  − 0.701). The composition of gut bacterial communities changed in a similar manner over time towards a common decay community. OTUs belonging to Bacteroidales (Bacteroides,Parabacteroides) significantly declined while Clostridiales (Clostridium,Anaerosphaera) and the fly-associated GammaproteobacteriaIgnatzschineriaandWohlfahrtiimonasincreased. Our examination of human caeca microflora in decomposing cadavers adds to the growing literature on postmortem microbial communities, which will ultimately contribute to a better understanding of decomposition processes.

    

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5. Cadaver Microbial Signatures of the Submandibular Glands and Thyroid for Forensic Investigations

   Sheree Finley, PhD* ; Matteo Moretti, MD ; Silvia Visonà, PhD ; Gulnaz Javan, PhD* ( January 2023 , Cadaver Microbial Signatures of the Submandibular Glands and Thyroid for Forensic Investigations)

   The study of the thyroid is an emerging topic, particularly in postmortem microbiome studies, due to the organ’s ability to affect the endocrine system. Also, the submandibular gland is a promising, emerging gland of study due to its position relative to the oral cavity. Previous thanatomicrobiome studies have demonstrated that bacteria belonging to the phyla Firmicutes, Proteobacteria, Bacteroides, and Pseudomonadota predominate internal organs and have been considered an important biomarker for postmortem interval. Further, Clostridium species that dominate in internal organs are linked to the hypoxic change that occurs after death, which leads to the switch of bacteria to become obligate anaerobes. Therefore, obligate anaerobes dominate the body after death due to their ability to thrive off fermentation products. 16S rRNA gene sequencing has been critical in thanatomicrobiome studies, which refers to the human microbiome (microorganisms within the body) after death. Currently, it has not been elucidated regarding the microorganisms that are associated with the decay of submandibular and thyroid glands. We hypothesized that through sequencing of the 16S rRNA gene of the submandibular and thyroid glands, the presence of Firmicutes and Proteobacteria will indicate potential biomarkers for postmortem interval. The present study revealed the postmortem microbial signatures of the submandibular and thyroid glands using the 16S rRNA gene, specifically the V3-V4 hypervariable regions, using universal primers 341F and 805R. We investigated a total of 37 cadavers obtained from ongoing criminal casework, 17 submandibular samples and 20 thyroid samples, and found that there is a correlation between microbial abundance in these postmortem glands. The predominating phyla of interest found in both glands were Firmicutes and Proteobacteria. The predominating genera were Paeniclostridium and Streptococcus in both glands, respectively. Further experimentation of the submandibular and thyroid glands will help to link oral thanatomicrobiome communities to “microbial clock” determinations, thus enhancing postmortem interval estimation. 

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