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The necrobiome has been defined as the community of species associated with decaying corpse remains.^[1] The process of decomposition is complex. Microbes decompose cadavers, but other organisms including fungi, nematodes, insects, and larger scavenger animals also contribute.^[2] Once the immune system is no longer active, microbes colonizing the intestines and lungs decompose their respective tissues and then travel throughout the body via the blood and lymphatic systems to break down other tissue and bone.^[3] During this process, gases are released as a by-product and accumulate, causing bloating.^[4] Eventually, the gases seep through the body’s wounds and natural openings, providing a way for some microbes to exit from the inside of the cadaver and inhabit the outside.^[3] The microbial communities colonizing the internal organs of a cadaver are referred to as the thanatomicrobiome.^[5] The region outside of the cadaver that is exposed to the external environment is referred to as the epinecrotic portion of the necrobiome,^[6]^[7]^[5] and is especially important when determining the time and location of death for an individual.^[6] Different microbes play specific roles during each stage of the decomposition process. The microbes that will colonize the cadaver and the rate of their activity are determined by the cadaver itself and the cadaver’s surrounding environmental conditions.^[7]^[8]

History

There is textual evidence that human cadavers were first studied around the third century BC to gain an understanding of human anatomy.^[9] Many of the first human cadaver studies took place in Italy, where the earliest record of determining the cause of death from a human corpse dates back to 1286.^[9] However, understanding of the human body progressed slowly, in part because the spread of Christianity and other religious beliefs resulted in human dissection becoming illegal.^[9] Thus, non-human animals were solely dissected for anatomical understanding until the 13th century when officials realized human cadavers were necessary for a better understanding of the human body.^[9] It was not until 1676 that Antonie van Leeuwenhoek designed a lens that made it possible to visualize microbes,^[10] and not until the late 18th century when microbes were considered useful in understanding the body after death.^[11] Modern sophisticated molecular techniques have made it possible to identify the microbial communities that inhabit and decompose cadavers, but more advanced research is fairly new, and therefore poorly understood.^[5] Studying the necrobiome has become increasingly useful in determining the time and cause of death.^[7]^[5] so more recent research can have crime-solving applications.

Necrobiome applications

Forensic entomology

Forensic entomology, the study of insects (arthropods) found in decomposing humans is the most popular field of study used in determining the post-mortem interval (PMI).^[8] This technique, however, is still new and consistently being improved, and--as such--it can work well with other techniques such as forensic anthropology, though forensic entomology is not as dependable on its own yet. Forensic entomologists often work within the field of crime scene investigations and are a part of the crime scene expert team that analyzes and collects evidence regarding a suspicious death. Typically, the minimum education required for this particular position is a Doctorate in Forensic Science. Forensic entomologists are experts in their field, and thus require a professional certification with the American Board of Forensic Entomology. As another relatively new field, forensic microbiologists, studying the presence of microbes, began investigating ways to determine time and place of death by analyzing the microbes present on the corpse.^[12] This would become an integral part of crime solving in later years upon the invention of the microbial clock process. The microbial timeline in which the body decays has been given the term “microbial clock,” this estimates how long a body has been in a certain place based on microbes present or missing.^[13] The succession of bacterial species populating the body after a period of four days is an indicator of minimum time since death (MTD).^[14] The presence or absence of maggots, as well as their age, can also be used to determine time of death; If the maggot is only a few days old, then the cadaver could not have been dead for longer than this time.^[15]

Microbial forensics

As the necrobiome deals with the various communities of bacteria and organisms that catalyze the decomposition of plants and animals (See Figure 1), this particular biome is an increasingly vital part of forensic science. The microbes occupying the space underneath and around a decomposing body are unique to it--similar to how fingerprints are exclusively unique to only one person.^[16] Using this differentiation, forensic investigators at a crime scene are able to distinguish between burial sites. This would provide concrete factual information about how long the body has been there and the predicted area in which the death possibly occurred.^[17] As the progression of research regarding microbial forensics and the necrobiome continues to be refined and improved, the need for new forensic scientists and microbiologists becomes increasingly necessary. When a crime such as murder has transpired, a team of crime scene specialists or forensic science experts are called to the scene to collect evidence and examine the body.^[18] These experts range from forensic odontologists to forensic microbiologists (See Figure 2). Together, they can obtain the necessary elements needed in order to properly reconstruct the victim’s demise.

Cadavers and Carcasses

One way many people study how bodies decompose is through the use of body farms. There are seven research facilities in the US that are home to body farms: University of Tennessee in Knoxville, Western Carolina University, Texas State University, Sam Houston State University, Southern Illinois University, Colorado Mesa University, and University of South Florida. These facilities study the decomposition of cadavers in all possible manners of decay, including in open or frozen environments, buried underground, or within cars.^[19] Through the study of the cadavers, experts examine the microbial timeline and document what’s normal in each stage in the various locations that each body is placed.^[20] An experiment was conducted to study the change in the necrobiome within a carcass and they performed it.^[21] The experiment was conducted to study the relative abundance of organisms in the necrobiome and the changes that occur during three different stages. For the experiment, they used six dead rabbits purchased from a pet food company. The rabbits were purchased from Kiezebank and exposed on top of a roof at the University of Huddersfield in West Yorkshire, United Kingdom. The rabbits were dead prior to purchase. Three of the rabbits’ fur were removed from the torso to identify any difference in necrobiome abundance. Samples were collected from inside of the mouth, the upper skin of the torso exposed to the air environment, and the bottom skin of the torso that’s touched by the soil. Active, advanced, and decay stages were examined, and proteobacteria were the most abundant present, followed by Firmicutes, Bacteroidetes, and Actinobacteria during the active stage of decomposition (Figure 3). During the advanced stage of decomposition, Proteobacteria decreased from 99.4% to 81.6% in the oral cavity but were most abundant in the non-fur samples. It was distinguished that Firmicutes were the most abundant for the skin samples in both fur and non-fur samples. Finally, Proteobacteria was most abundant in the soil interface during the beginning of decomposition in both fur and non-fur samples. Also, they noted that Actinobacteria was the least abundant in the active stage and decreased even more during the dry stage.^[21]

Decomposition

The manner in which bacteria colonize a cadaver is predictable when examining the time since death.^[22] Only recent studies have taken place to determine if bacteria alone can inform the postmortem interval.^[23] Bacteria responsible for decomposing cadavers can be difficult to study because the bacteria found on a cadaver varies and changes quickly.^[24]^[23] Bacteria can be brought to a cadaver by scavengers, air, or water.^[25] Other environmental factors like temperature and soil can impact the microbes found on a cadaver.^[25] Fortunately, microbial colonization between humans and animals is so similar, that animal models can be used to understand the decomposition process for humans.^[26] Human cadavers are used for research, but animal models provide larger sample sizes and produce more controlled studies.^[23]^[22] Swine models have been used repeatedly to understand the human decomposition process in terrestrial environments.^[27]^[28] Swine are suitable for studying human decomposition because of their size, sparse hairs, and similar bacteria found in their GI tracts.^[29]

Technology and techniques

An algorithm has been developed to accurately predict time since death with an accuracy of within two days.^[30] This multi-step process focuses on criminal investigations and forensics science to obtain the post-mortem interval. Before the continuous development, there was no universal algorithm. Algorithms are complicated due to various factors in both biotic and abiotic which leads to a bimolecular process.^[8] One change can make a dramatic difference to the necrobiome (i.e. body mass index, the clothing on the carcass, body temperature, contact with the ground).

It is difficult to come to an agreement on technology and technique because of different rules for each region. They suggested that because of genetic variation, DNA profiling is the best approach; however, algorithms developed within the United States are not applicable to other regions due to environmental variations. Currently, the majority of algorithms use genomic analysis for understanding microbial species that inhabit soil and their contribution to the decomposition process.^[8]Techniques for analyzing the necrobiome have now been coupled with forensic entomology, such as phospholipid fatty acid (PLFA) analysis,^[31] total soil fatty acid methyl esters,^[31] and DNA profiling.^[31] Pig carcasses have also become a tool to understand human microbiology, minimizing the issue of variation that exists when using human cadavers as study subjects.^[31] This technology is used to simplify the sample collection into sequences that scientists can read. The simplified sequence of the necrobiome is run through a data bank to match the name of it. Due to the lack of universal algorithm technology, there is a knowledge gap in various platforms across different regions of the world. In order to close that gap, there needs to be an expansion of the technology. However, there are a few obstacles, including identifying needs, research, prototype development, acceptance, and adoption.^[32] Overcoming these obstacles would assist many organizations that are involved with forensic science. Also, it would increase the understanding of the necrobiome and growth of developing a successful accurate multi-step experiment. The samples are loaded into a machine to generate and analyze DNA sequences of the microbiome. Algorithms are done in a lab on a computer program to read and match the sequences within the data bank. The results return very quickly within a few minutes to the latest days.

Conclusion

The necrobiome is becoming more understood everyday. New technology and techniques have made this possible. A considerable amount of research on the necrobiome contributes to obtaining an accurate time of death. Forensic entomology and microbiology, though not yet dependable on their own, are useful when applied in conjunction with one another other. Although it is clear that microbes play an important role in the decomposition process, they are poorly studied due to the inconsistencies between cadavers. However, research continues to advance. The use of animal models has contributed to providing more robust research that can be replicated, and human cadavers are now studied under various environmental conditions at body farms. Algorithms have also been created to help determine the time of death.

References

^ Benbow ME, Lewis AJ, Tomberlin JK, Pechal JL (March 2013). "Seasonal necrophagous insect community assembly during vertebrate carrion decomposition". Journal of Medical Entomology. 50 (2): 440–50. doi:10.1603/me12194. PMID 23540134.
^ Yong, Ed (2015-12-10). "Meet the Necrobiome: The Microbes That Will Eat Your Corpse". The Atlantic. Retrieved 2020-04-28. {{cite web}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ ^a ^b Janaway RC (1996). "The decay of buried human remains and their associated materials.". In Hunter J, Roberts C, Martin A (eds.). Studies in Crime: An Introduction to Forensic Archaeology. London: Batsford. pp. 58–85.
^ Vass AA, Barshick SA, Sega G, Caton J, Skeen JT, Love JC, Synstelien JA (May 2002). "Decomposition chemistry of human remains: a new methodology for determining the postmortem interval". Journal of Forensic Sciences. 47 (3): 542–53. PMID 12051334.
^ ^a ^b ^c ^d Ventura Spagnolo E, Stassi C, Mondello C, Zerbo S, Milone L, Argo A (February 2019). "Forensic microbiology applications: A systematic review". Legal Medicine. 36: 73–80. doi:10.1016/j.legalmed.2018.11.002. PMID 30419494.
^ ^a ^b Zhou W, Bian Y (2018-04-03). "Thanatomicrobiome composition profiling as a tool for forensic investigation". Forensic Sciences Research. 3 (2): 105–110. doi:10.1080/20961790.2018.1466430. PMC 6197100. PMID 30483658.
^ ^a ^b ^c Javan GT, Finley SJ, Can I, Wilkinson JE, Hanson JD, Tarone AM (July 2016). "Human Thanatomicrobiome Succession and Time Since Death". Scientific Reports. 6 (1): 29598. doi:10.1038/srep29598. PMC 4944132. PMID 27412051.
^ ^a ^b ^c ^d Abdelhalim K, Khaoula B, Saddek B, Abbas M, Mokhtar HC (2017). "Post Mortem Interval: Necrobiome Analysis Using Artificial Neural Networks". Computational Biology and Bioinformatics. 5 (6): 90. doi:10.11648/j.cbb.20170506.13.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ ^a ^b ^c ^d Ghosh SK (September 2015). "Human cadaveric dissection: a historical account from ancient Greece to the modern era". Anatomy & Cell Biology. 48 (3): 153–69. doi:10.5115/acb.2015.48.3.153. PMC 4582158. PMID 26417475.
^ Young, Ed (2016). I contain multitudes: the microbes within us and a grander view of life. 195 Broadway, New York, NY 10007: HarperCollins Publishers. ISBN 978-0-06-236860-7. {{cite book}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)CS1 maint: location (link)
^ Riedel S (April 2014). "The value of postmortem microbiology cultures". Journal of Clinical Microbiology. 52 (4): 1028–33. doi:10.1128/JCM.03102-13. PMC 3993482. PMID 24403308.
^ Lehman, Donald C. (April 2014). "Forensic Microbiology". Clinical Microbiology Newsletter. 36 (7): 49–54. doi:10.1016/j.clinmicnews.2014.03.001. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ Metcalf JL, Wegener Parfrey L, Gonzalez A, Lauber CL, Knights D, Ackermann G, et al. (October 2013). "A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system". eLife. 2: e01104. doi:10.7554/eLife.01104. PMID 24137541.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ Hauther KA, Cobaugh KL, Jantz LM, Sparer TE, DeBruyn JM (September 2015). "Estimating Time Since Death from Postmortem Human Gut Microbial Communities". Journal of Forensic Sciences. 60 (5): 1234–40. doi:10.1111/1556-4029.12828. PMID 26096156.
^ Erzinçlioglu Z (1 January 2003). "Forensic entomology". Clinical Medicine. 3 (1): 74–6. doi:10.7861/clinmedicine.3-1-74. PMID 12617420.
^ Franzosa EA, Huang K, Meadow JF, Gevers D, Lemon KP, Bohannan BJ, Huttenhower C (June 2015). "Identifying personal microbiomes using metagenomic codes". Proceedings of the National Academy of Sciences of the United States of America. 112 (22): E2930-8. doi:10.1073/pnas.1423854112. PMID 25964341.
^ Benbow ME, Lewis AJ, Tomberlin JK, Pechal JL (March 2013). "Seasonal necrophagous insect community assembly during vertebrate carrion decomposition". Journal of Medical Entomology. 50 (2): 440–50. doi:10.1603/me12194. PMID 23540134.
^ Lutui, Raymond (November 2016). "A multidisciplinary digital forensic investigation process model". Business Horizons. 59 (6): 593–604. doi:10.1016/j.bushor.2016.08.001. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ Wallman JF (December 2017). "Body farms". Forensic Science, Medicine, and Pathology. 13 (4): 487–489. doi:10.1007/s12024-017-9932-z. PMID 29075978.
^ Wallman JF (December 2017). "Body farms". Forensic Science, Medicine, and Pathology. 13 (4): 487–489. doi:10.1007/s12024-017-9932-z. PMID 29075978.
^ ^a ^b Tuccia F, Zurgani E, Bortolini S, Vanin S (September 2019). "Experimental evaluation on the applicability of necrobiome analysis in forensic veterinary science". MicrobiologyOpen. 8 (9): e00828. doi:10.1002/mbo3.828. PMC 6741123. PMID 30861327.
^ ^a ^b Finley SJ, Benbow ME, Javan GT (May 2015). "Microbial communities associated with human decomposition and their potential use as postmortem clocks". International Journal of Legal Medicine. 129 (3): 623–32. doi:10.1007/s00414-014-1059-0. PMID 25129823.
^ ^a ^b ^c Hyde ER, Metcalf JL, Bucheli SR, Lynne AM, Knight R (2017). "Microbial communities associated with decomposing corpses". Forensic Microbiology. John Wiley & Sons, Ltd: 245–273. doi:10.1002/9781119062585.ch10. ISBN 978-1-119-06258-5.
^ Vass A (2001). "Beyond the grave—understanding human decomposition". Microbiology Today. 28 (28): 190–192.
^ ^a ^b Hyde ER, Haarmann DP, Lynne AM, Bucheli SR, Petrosino JF (2013-10-30). "The living dead: bacterial community structure of a cadaver at the onset and end of the bloat stage of decomposition". PloS One. 8 (10): e77733. doi:10.1371/journal.pone.0077733. PMC 3813760. PMID 24204941.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ Burcham ZM, Hood JA, Pechal JL, Krausz KL, Bose JL, Schmidt CJ, et al. (July 2016). "Fluorescently labeled bacteria provide insight on post-mortem microbial transmigration". Forensic Science International. 264: 63–9. doi:10.1016/j.forsciint.2016.03.019. PMID 27032615.
^ Carter DO, Metcalf JL, Bibat A, Knight R (June 2015). "Seasonal variation of postmortem microbial communities". Forensic Science, Medicine, and Pathology. 11 (2): 202–7. doi:10.1007/s12024-015-9667-7. PMID 25737335.
^ Pechal JL, Crippen TL, Tarone AM, Lewis AJ, Tomberlin JK, Benbow ME (2013-11-12). "Microbial community functional change during vertebrate carrion decomposition". PloS One. 8 (11): e79035. doi:10.1371/journal.pone.0079035. PMID 24265741.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ Schoenly KG, Haskell NH, Mills DK, Bieme-Ndi C, Larsen K, Lee Y (2006-09-01). "Recreating Death's Acre in the School Yard: Using Pig Carcasses as Model Corpses, to Teach Concepts of Forensic Entomology & Ecological Succession". The American Biology Teacher. 68 (7): 402–410. doi:10.2307/4452028.
^ Johnson HR, Trinidad DD, Guzman S, Khan Z, Parziale JV, DeBruyn JM, Lents NH (2016). "A Machine Learning Approach for Using the Postmortem Skin Microbiome to Estimate the Postmortem Interval". Plos One. 11 (12): e0167370. doi:10.1371/journal.pone.0167370. PMC 5179130. PMID 28005908.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ ^a ^b ^c ^d Parkinson RA, Dias KR, Horswell J, Greenwood P, Banning N, Tibbett M, Vass AA (2009). "Microbial community analysis of human decomposition on soil.". Criminal and environmental soil forensics. Dordrecht: Springer. pp. 379–394. doi:10.1007/978-1-4020-9204-6_24.
^ Metcalf JL (January 2019). "Estimating the postmortem interval using microbes: Knowledge gaps and a path to technology adoption". Forensic Science International. Genetics. 38: 211–218. doi:10.1016/j.fsigen.2018.11.004. PMID 30448529.

[1] Benbow ME, Lewis AJ, Tomberlin JK, Pechal JL (March 2013). "Seasonal necrophagous insect community assembly during vertebrate carrion decomposition". Journal of Medical Entomology. 50 (2): 440–50. doi:10.1603/me12194. PMID 23540134.

[2] Yong, Ed (2015-12-10). "Meet the Necrobiome: The Microbes That Will Eat Your Corpse". The Atlantic. Retrieved 2020-04-28. {{cite web}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)

[:8-3] Janaway RC (1996). "The decay of buried human remains and their associated materials.". In Hunter J, Roberts C, Martin A (eds.). Studies in Crime: An Introduction to Forensic Archaeology. London: Batsford. pp. 58–85.

[pmid12051334-4] Vass AA, Barshick SA, Sega G, Caton J, Skeen JT, Love JC, Synstelien JA (May 2002). "Decomposition chemistry of human remains: a new methodology for determining the postmortem interval". Journal of Forensic Sciences. 47 (3): 542–53. PMID 12051334.

[:0-5] Ventura Spagnolo E, Stassi C, Mondello C, Zerbo S, Milone L, Argo A (February 2019). "Forensic microbiology applications: A systematic review". Legal Medicine. 36: 73–80. doi:10.1016/j.legalmed.2018.11.002. PMID 30419494.

[:3-6] Zhou W, Bian Y (2018-04-03). "Thanatomicrobiome composition profiling as a tool for forensic investigation". Forensic Sciences Research. 3 (2): 105–110. doi:10.1080/20961790.2018.1466430. PMC 6197100. PMID 30483658.

[:1-7] Javan GT, Finley SJ, Can I, Wilkinson JE, Hanson JD, Tarone AM (July 2016). "Human Thanatomicrobiome Succession and Time Since Death". Scientific Reports. 6 (1): 29598. doi:10.1038/srep29598. PMC 4944132. PMID 27412051.

[:2-8] Abdelhalim K, Khaoula B, Saddek B, Abbas M, Mokhtar HC (2017). "Post Mortem Interval: Necrobiome Analysis Using Artificial Neural Networks". Computational Biology and Bioinformatics. 5 (6): 90. doi:10.11648/j.cbb.20170506.13.{{cite journal}}: CS1 maint: unflagged free DOI (link)

[:4-9] Ghosh SK (September 2015). "Human cadaveric dissection: a historical account from ancient Greece to the modern era". Anatomy & Cell Biology. 48 (3): 153–69. doi:10.5115/acb.2015.48.3.153. PMC 4582158. PMID 26417475.

[10] Young, Ed (2016). I contain multitudes: the microbes within us and a grander view of life. 195 Broadway, New York, NY 10007: HarperCollins Publishers. ISBN 978-0-06-236860-7. {{cite book}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)CS1 maint: location (link)

[11] Riedel S (April 2014). "The value of postmortem microbiology cultures". Journal of Clinical Microbiology. 52 (4): 1028–33. doi:10.1128/JCM.03102-13. PMC 3993482. PMID 24403308.

[12] Lehman, Donald C. (April 2014). "Forensic Microbiology". Clinical Microbiology Newsletter. 36 (7): 49–54. doi:10.1016/j.clinmicnews.2014.03.001. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)

[13] Metcalf JL, Wegener Parfrey L, Gonzalez A, Lauber CL, Knights D, Ackermann G, et al. (October 2013). "A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system". eLife. 2: e01104. doi:10.7554/eLife.01104. PMID 24137541.{{cite journal}}: CS1 maint: unflagged free DOI (link)

[14] Hauther KA, Cobaugh KL, Jantz LM, Sparer TE, DeBruyn JM (September 2015). "Estimating Time Since Death from Postmortem Human Gut Microbial Communities". Journal of Forensic Sciences. 60 (5): 1234–40. doi:10.1111/1556-4029.12828. PMID 26096156.

[15] Erzinçlioglu Z (1 January 2003). "Forensic entomology". Clinical Medicine. 3 (1): 74–6. doi:10.7861/clinmedicine.3-1-74. PMID 12617420.

[16] Franzosa EA, Huang K, Meadow JF, Gevers D, Lemon KP, Bohannan BJ, Huttenhower C (June 2015). "Identifying personal microbiomes using metagenomic codes". Proceedings of the National Academy of Sciences of the United States of America. 112 (22): E2930-8. doi:10.1073/pnas.1423854112. PMID 25964341.

[17] Benbow ME, Lewis AJ, Tomberlin JK, Pechal JL (March 2013). "Seasonal necrophagous insect community assembly during vertebrate carrion decomposition". Journal of Medical Entomology. 50 (2): 440–50. doi:10.1603/me12194. PMID 23540134.

[18] Lutui, Raymond (November 2016). "A multidisciplinary digital forensic investigation process model". Business Horizons. 59 (6): 593–604. doi:10.1016/j.bushor.2016.08.001. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)

[19] Wallman JF (December 2017). "Body farms". Forensic Science, Medicine, and Pathology. 13 (4): 487–489. doi:10.1007/s12024-017-9932-z. PMID 29075978.

[20] Wallman JF (December 2017). "Body farms". Forensic Science, Medicine, and Pathology. 13 (4): 487–489. doi:10.1007/s12024-017-9932-z. PMID 29075978.

[pmid30861327-21] Tuccia F, Zurgani E, Bortolini S, Vanin S (September 2019). "Experimental evaluation on the applicability of necrobiome analysis in forensic veterinary science". MicrobiologyOpen. 8 (9): e00828. doi:10.1002/mbo3.828. PMC 6741123. PMID 30861327.

[:7-22] Finley SJ, Benbow ME, Javan GT (May 2015). "Microbial communities associated with human decomposition and their potential use as postmortem clocks". International Journal of Legal Medicine. 129 (3): 623–32. doi:10.1007/s00414-014-1059-0. PMID 25129823.

[:5-23] Hyde ER, Metcalf JL, Bucheli SR, Lynne AM, Knight R (2017). "Microbial communities associated with decomposing corpses". Forensic Microbiology. John Wiley & Sons, Ltd: 245–273. doi:10.1002/9781119062585.ch10. ISBN 978-1-119-06258-5.

[24] Vass A (2001). "Beyond the grave—understanding human decomposition". Microbiology Today. 28 (28): 190–192.

[:6-25] Hyde ER, Haarmann DP, Lynne AM, Bucheli SR, Petrosino JF (2013-10-30). "The living dead: bacterial community structure of a cadaver at the onset and end of the bloat stage of decomposition". PloS One. 8 (10): e77733. doi:10.1371/journal.pone.0077733. PMC 3813760. PMID 24204941.{{cite journal}}: CS1 maint: unflagged free DOI (link)

[26] Burcham ZM, Hood JA, Pechal JL, Krausz KL, Bose JL, Schmidt CJ, et al. (July 2016). "Fluorescently labeled bacteria provide insight on post-mortem microbial transmigration". Forensic Science International. 264: 63–9. doi:10.1016/j.forsciint.2016.03.019. PMID 27032615.

[27] Carter DO, Metcalf JL, Bibat A, Knight R (June 2015). "Seasonal variation of postmortem microbial communities". Forensic Science, Medicine, and Pathology. 11 (2): 202–7. doi:10.1007/s12024-015-9667-7. PMID 25737335.

[28] Pechal JL, Crippen TL, Tarone AM, Lewis AJ, Tomberlin JK, Benbow ME (2013-11-12). "Microbial community functional change during vertebrate carrion decomposition". PloS One. 8 (11): e79035. doi:10.1371/journal.pone.0079035. PMID 24265741.{{cite journal}}: CS1 maint: unflagged free DOI (link)

[29] Schoenly KG, Haskell NH, Mills DK, Bieme-Ndi C, Larsen K, Lee Y (2006-09-01). "Recreating Death's Acre in the School Yard: Using Pig Carcasses as Model Corpses, to Teach Concepts of Forensic Entomology & Ecological Succession". The American Biology Teacher. 68 (7): 402–410. doi:10.2307/4452028.

[pmid28005908-30] Johnson HR, Trinidad DD, Guzman S, Khan Z, Parziale JV, DeBruyn JM, Lents NH (2016). "A Machine Learning Approach for Using the Postmortem Skin Microbiome to Estimate the Postmortem Interval". Plos One. 11 (12): e0167370. doi:10.1371/journal.pone.0167370. PMC 5179130. PMID 28005908.{{cite journal}}: CS1 maint: unflagged free DOI (link)

[Parkinson_2009-31] Parkinson RA, Dias KR, Horswell J, Greenwood P, Banning N, Tibbett M, Vass AA (2009). "Microbial community analysis of human decomposition on soil.". Criminal and environmental soil forensics. Dordrecht: Springer. pp. 379–394. doi:10.1007/978-1-4020-9204-6_24.

[pmid30448529-32] Metcalf JL (January 2019). "Estimating the postmortem interval using microbes: Knowledge gaps and a path to technology adoption". Forensic Science International. Genetics. 38: 211–218. doi:10.1016/j.fsigen.2018.11.004. PMID 30448529.

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-The '''necrobiome''' has been defined as the community of species associated with decaying corpse remains<ref>{{Cite journal|last=Benbow|first=M. E.|last2=Lewis|first2=A. J.|last3=Tomberlin|first3=J. K.|last4=Pechal|first4=J. L.|date=2013-03-01|title=Seasonal Necrophagous Insect Community Assembly During Vertebrate Carrion Decomposition|url=http://dx.doi.org/10.1603/me12194|journal=Journal of Medical Entomology|volume=50|issue=2|pages=440–450|doi=10.1603/me12194|issn=0022-2585}}</ref>. The process of decomposition is complex. Microbes decompose cadavers, but other organisms including fungi, nematodes, insects, and larger scavenger animals also contribute<ref>{{Cite web|title=Meet the Necrobiome: The Microbes That Will Eat Your Corpse|url=https://www.theatlantic.com/science/archive/2015/12/meet-the-necrobiome-the-predictable-microbes-that-will-eat-your-dying-corpse/419676/|last=Yong|first=Ed|date=2015-12-10|website=The Atlantic|language=en-US|access-date=2020-04-28}}</ref>. Once the immune system is no longer active, microbes colonizing the intestines and lungs decompose their respective tissues and then travel throughout the body via the blood and lymphatic systems to break down other tissue and bone<ref name=":8">Janaway RC. The decay of buried human remains and their associated materials. In J Hunter, C Roberts and A Martin eds. Studies in Crime: An Introduction to Forensic Archaeology. London: Batsford, pp. 58–85. 1996.</ref>. During this process, gases are released as a by-product and accumulate, causing bloating<ref>Vass, A., Barshick, S., Sega, G., Caton, J., Skeen, J., Love, J., and Synstelien, J., "Decomposition Chemistry of Human Remains: A New Methodology for Determining the Postmortem Interval," ''Journal of Forensic Sciences'', Vol. 47, No. 3, 2002, pp. 542-553, ISSN 0022-1198</ref>. Eventually, the gases seep through the body’s wounds and natural openings, providing a way for some microbes to exit from the inside of the cadaver and inhabit the outside<ref name=":8" />. The microbial communities colonizing the internal organs of a cadaver are referred to as the thanatomicrobiome<ref name=":0">{{Cite journal|last=Ventura Spagnolo|first=Elvira|last2=Stassi|first2=Chiara|last3=Mondello|first3=Cristina|last4=Zerbo|first4=Stefania|last5=Milone|first5=Livio|last6=Argo|first6=Antonina|date=2019-02|title=Forensic microbiology applications: A systematic review|url=https://linkinghub.elsevier.com/retrieve/pii/S1344622318301536|journal=Legal Medicine|language=en|volume=36|pages=73–80|doi=10.1016/j.legalmed.2018.11.002}}</ref>. The region outside of the cadaver that is exposed to the external environment is referred to as the epinecrotic portion of the necrobiome<ref name=":3">{{Cite journal|last=Zhou|first=Wei|last2=Bian|first2=Yingnan|date=2018-04-03|title=Thanatomicrobiome composition profiling as a tool for forensic investigation|url=https://doi.org/10.1080/20961790.2018.1466430|journal=Forensic Sciences Research|volume=3|issue=2|pages=105–110|doi=10.1080/20961790.2018.1466430|issn=2096-1790|pmc=PMC6197100|pmid=30483658}}</ref><ref name=":1">{{Cite journal|last=Javan|first=Gulnaz T.|last2=Finley|first2=Sheree J.|last3=Can|first3=Ismail|last4=Wilkinson|first4=Jeremy E.|last5=Hanson|first5=J. Delton|last6=Tarone|first6=Aaron M.|date=2016-07-14|title=Human Thanatomicrobiome Succession and Time Since Death|url=https://www.nature.com/articles/srep29598|journal=Scientific Reports|language=en|volume=6|issue=1|pages=1–9|doi=10.1038/srep29598|issn=2045-2322|pmc=PMC4944132|pmid=27412051}}</ref><ref name=":0" />, and is especially important when determining the time and location of death for an individual<ref name=":3" />. Different microbes play specific roles during each stage of the decomposition process. The microbes that will colonize the cadaver and the rate of their activity are determined by the cadaver itself and the cadaver’s surrounding environmental conditions<ref name=":1" /><ref name=":2">{{cite journal|last1=Abdelhalim|first1=Khenchouche|date=2017|title=Post Mortem Interval: Necrobiome Analysis Using Artificial Neural Networks|journal=Computational Biology and Bioinformatics|volume=5|issue=6|pages=90|doi=10.11648/j.cbb.20170506.13}}</ref>.
+The '''necrobiome''' has been defined as the community of species associated with decaying corpse remains.<ref>{{cite journal | vauthors = Benbow ME, Lewis AJ, Tomberlin JK, Pechal JL | title = Seasonal necrophagous insect community assembly during vertebrate carrion decomposition | journal = Journal of Medical Entomology | volume = 50 | issue = 2 | pages = 440–50 | date = March 2013 | pmid = 23540134 | doi = 10.1603/me12194 | url = http://dx.doi.org/10.1603/me12194 }}</ref> The process of decomposition is complex. Microbes decompose cadavers, but other organisms including fungi, nematodes, insects, and larger scavenger animals also contribute.<ref>{{Cite web|title=Meet the Necrobiome: The Microbes That Will Eat Your Corpse |url= https://www.theatlantic.com/science/archive/2015/12/meet-the-necrobiome-the-predictable-microbes-that-will-eat-your-dying-corpse/419676/ |last=Yong |first=Ed | name-list-format = vanc |date=2015-12-10 |website=The Atlantic|language=en-US|access-date=2020-04-28}}</ref> Once the immune system is no longer active, microbes colonizing the intestines and lungs decompose their respective tissues and then travel throughout the body via the blood and lymphatic systems to break down other tissue and bone.<ref name=":8">{{cite book | vauthors = Janaway RC | chapter = The decay of buried human remains and their associated materials. | veditors = Hunter J, Roberts C, Martin A | title = Studies in Crime: An Introduction to Forensic Archaeology. | location = London | publisher = Batsford | pages = 58–85 | date = 1996 }}</ref> During this process, gases are released as a by-product and accumulate, causing bloating.<ref name="pmid12051334">{{cite journal | vauthors = Vass AA, Barshick SA, Sega G, Caton J, Skeen JT, Love JC, Synstelien JA | title = Decomposition chemistry of human remains: a new methodology for determining the postmortem interval | journal = Journal of Forensic Sciences | volume = 47 | issue = 3 | pages = 542–53 | date = May 2002 | pmid = 12051334 | doi = | url = }}</ref> Eventually, the gases seep through the body’s wounds and natural openings, providing a way for some microbes to exit from the inside of the cadaver and inhabit the outside.<ref name=":8" /> The microbial communities colonizing the internal organs of a cadaver are referred to as the thanatomicrobiome.<ref name=":0">{{cite journal | vauthors = Ventura Spagnolo E, Stassi C, Mondello C, Zerbo S, Milone L, Argo A | title = Forensic microbiology applications: A systematic review | journal = Legal Medicine | volume = 36 | pages = 73–80 | date = February 2019 | pmid = 30419494 | doi = 10.1016/j.legalmed.2018.11.002 }}</ref> The region outside of the cadaver that is exposed to the external environment is referred to as the epinecrotic portion of the necrobiome,<ref name=":3">{{cite journal | vauthors = Zhou W, Bian Y | title = Thanatomicrobiome composition profiling as a tool for forensic investigation | journal = Forensic Sciences Research | volume = 3 | issue = 2 | pages = 105–110 | date = 2018-04-03 | pmid = 30483658 | pmc = 6197100 | doi = 10.1080/20961790.2018.1466430 | url = https://doi.org/10.1080/20961790.2018.1466430 }}</ref><ref name=":1">{{cite journal | vauthors = Javan GT, Finley SJ, Can I, Wilkinson JE, Hanson JD, Tarone AM | title = Human Thanatomicrobiome Succession and Time Since Death | journal = Scientific Reports | volume = 6 | issue = 1 | pages = 29598 | date = July 2016 | pmid = 27412051 | pmc = 4944132 | doi = 10.1038/srep29598 | url = https://www.nature.com/articles/srep29598 }}</ref><ref name=":0" /> and is especially important when determining the time and location of death for an individual.<ref name=":3" /> Different microbes play specific roles during each stage of the decomposition process. The microbes that will colonize the cadaver and the rate of their activity are determined by the cadaver itself and the cadaver’s surrounding environmental conditions.<ref name=":1" /><ref name=":2">{{cite journal| vauthors = Abdelhalim K, Khaoula B, Saddek B, Abbas M, Mokhtar HC |date=2017|title=Post Mortem Interval: Necrobiome Analysis Using Artificial Neural Networks|journal=Computational Biology and Bioinformatics |volume=5 |issue=6 |pages=90 |doi=10.11648/j.cbb.20170506.13 }}</ref>
 === History ===
-There is textual evidence that human cadavers were first studied around the third century BC to gain an understanding of human anatomy<ref name=":4">{{Cite journal|last=Ghosh|first=Sanjib Kumar|date=2015|title=Human cadaveric dissection: a historical account from ancient Greece to the modern era|url=https://doi.org/10.5115/acb.2015.48.3.153|journal=Anatomy & Cell Biology|volume=48|issue=3|pages=153|doi=10.5115/acb.2015.48.3.153|issn=2093-3665|pmc=PMC4582158|pmid=26417475}}</ref>. Many of the first human cadaver studies took place in Italy, where the earliest record of determining the cause of death from a human corpse dates back to 1286<ref name=":4" />. However, understanding of the human body progressed slowly, in part because the spread of Christianity and other religious beliefs resulted in human dissection becoming illegal<ref name=":4" />. Thus, non-human animals were solely dissected for anatomical understanding until the 13th century when officials realized human cadavers were necessary for a better understanding of the human body<ref name=":4" />. It was not until 1676 that Antonie van Leeuwenhoek designed a lens that made it possible to visualize microbes<ref>{{Cite book|last=Young|first=Ed|title=I contain multitudes: the microbes within us and a grander view of life|publisher=HarperCollins Publishers|year=2016|isbn=978-0-06-236860-7|location=195 Broadway, New York, NY 10007|pages=}}</ref>, and not until the late 18th century when microbes were considered useful in understanding the body after death<ref>{{Cite journal|last=Riedel|first=Stefan|date=2014-04-01|title=The Value of Postmortem Microbiology Cultures|url=https://jcm.asm.org/content/52/4/1028|journal=Journal of Clinical Microbiology|language=en|volume=52|issue=4|pages=1028–1033|doi=10.1128/JCM.03102-13|issn=0095-1137|pmc=PMC3993482|pmid=24403308}}</ref>. Modern sophisticated molecular techniques have made it possible to identify the microbial communities that inhabit and decompose cadavers, but more advanced research is fairly new, and therefore poorly understood<ref name=":0" />. Studying the necrobiome has become increasingly useful in determining the time and cause of death<ref name=":1" /><ref name=":0" />, so more recent research can have crime-solving applications.
+There is textual evidence that human cadavers were first studied around the third century BC to gain an understanding of human anatomy.<ref name=":4">{{cite journal | vauthors = Ghosh SK | title = Human cadaveric dissection: a historical account from ancient Greece to the modern era | journal = Anatomy & Cell Biology | volume = 48 | issue = 3 | pages = 153–69 | date = September 2015 | pmid = 26417475 | pmc = 4582158 | doi = 10.5115/acb.2015.48.3.153 }}</ref> Many of the first human cadaver studies took place in Italy, where the earliest record of determining the cause of death from a human corpse dates back to 1286.<ref name=":4" /> However, understanding of the human body progressed slowly, in part because the spread of Christianity and other religious beliefs resulted in human dissection becoming illegal.<ref name=":4" /> Thus, non-human animals were solely dissected for anatomical understanding until the 13th century when officials realized human cadavers were necessary for a better understanding of the human body.<ref name=":4" /> It was not until 1676 that Antonie van Leeuwenhoek designed a lens that made it possible to visualize microbes,<ref>{{Cite book|last=Young|first=Ed | name-list-format = vanc |title=I contain multitudes: the microbes within us and a grander view of life|publisher=HarperCollins Publishers|year=2016|isbn=978-0-06-236860-7|location=195 Broadway, New York, NY 10007|pages=}}</ref> and not until the late 18th century when microbes were considered useful in understanding the body after death.<ref>{{cite journal | vauthors = Riedel S | title = The value of postmortem microbiology cultures | journal = Journal of Clinical Microbiology | volume = 52 | issue = 4 | pages = 1028–33 | date = April 2014 | pmid = 24403308 | pmc = 3993482 | doi = 10.1128/JCM.03102-13 | url = https://jcm.asm.org/content/52/4/1028 }}</ref> Modern sophisticated molecular techniques have made it possible to identify the microbial communities that inhabit and decompose cadavers, but more advanced research is fairly new, and therefore poorly understood.<ref name=":0" /> Studying the necrobiome has become increasingly useful in determining the time and cause of death.<ref name=":1" /><ref name=":0" /> so more recent research can have crime-solving applications.
-=== Necrobiome Applications ===
+=== Necrobiome applications ===
-==== Forensic Entomology ====
+==== Forensic entomology ====
-Forensic entomology, the study of insects (arthropods) found in decomposing humans is the most popular field of study used in determining the post-mortem interval (PMI)<ref name=":2" />.  This technique, however, is still new and consistently being improved, and--as such--it can work well with other techniques such as forensic anthropology, though forensic entomology is not as dependable on its own yet. Forensic entomologists often work within the field of crime scene investigations and are a part of the crime scene expert team that analyzes and collects evidence regarding a suspicious death. Typically, the minimum education required for this particular position is a Doctorate in Forensic Science. Forensic entomologists are experts in their field, and thus require a professional certification with the American Board of Forensic Entomology. As another relatively new field, forensic microbiologists, studying the presence of microbes, began investigating ways to determine time and place of death by analyzing the microbes present on the corpse <ref>{{cite journal|last1=Lehman|first1=Donald C.|date=April 2014|title=Forensic Microbiology|journal=Clinical Microbiology Newsletter|volume=36|issue=7|pages=49–54|doi=10.1016/j.clinmicnews.2014.03.001}}</ref>. This would become an integral part of crime solving in later years upon the invention of the microbial clock process.  The microbial timeline in which the body decays has been given the term “microbial clock,” this estimates how long a body has been in a certain place based on microbes present or missing <ref>{{cite journal|last1=Metcalf|first1=Jessica L|last2=Wegener Parfrey|first2=Laura|last3=Gonzalez|first3=Antonio|last4=Lauber|first4=Christian L|last5=Knights|first5=Dan|last6=Ackermann|first6=Gail|last7=Humphrey|first7=Gregory C|last8=Gebert|first8=Matthew J|last9=Van Treuren|first9=Will|last10=Berg-Lyons|first10=Donna|last11=Keepers|first11=Kyle|date=15 October 2013|title=A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system|journal=eLife|volume=2|doi=10.7554/eLife.01104|first14=Noah|last14=Fierer|first15=David O|last16=Knight|first16=Rob|first13=James|last13=Bullard|first12=Yan|last12=Guo|last15=Carter}}</ref>.The succession of bacterial species populating the body after a period of four days is an indicator of minimum time since death (MTD) <ref>{{cite journal|last1=Hauther|first1=Kathleen A.|last2=Cobaugh|first2=Kelly L.|last3=Jantz|first3=Lee Meadows|last4=Sparer|first4=Tim E.|last5=DeBruyn|first5=Jennifer M.|date=September 2015|title=Estimating Time Since Death from Postmortem Human Gut Microbial Communities|journal=Journal of Forensic Sciences|volume=60|issue=5|pages=1234–1240|doi=10.1111/1556-4029.12828}}</ref>. The presence or absence of maggots, as well as their age, can also be used to determine time of death; If the maggot is only a few days old, then the cadaver could not have been dead for longer than this time <ref>{{cite journal|last1=Erzinçlioglu|first1=Zakaria|date=1 January 2003|title=Forensic entomology|journal=Clinical Medicine|volume=3|issue=1|pages=74–76|doi=10.7861/clinmedicine.3-1-74}}</ref>.
+Forensic entomology, the study of insects (arthropods) found in decomposing humans is the most popular field of study used in determining the post-mortem interval (PMI).<ref name=":2" />  This technique, however, is still new and consistently being improved, and--as such--it can work well with other techniques such as forensic anthropology, though forensic entomology is not as dependable on its own yet. Forensic entomologists often work within the field of crime scene investigations and are a part of the crime scene expert team that analyzes and collects evidence regarding a suspicious death. Typically, the minimum education required for this particular position is a Doctorate in Forensic Science. Forensic entomologists are experts in their field, and thus require a professional certification with the American Board of Forensic Entomology. As another relatively new field, forensic microbiologists, studying the presence of microbes, began investigating ways to determine time and place of death by analyzing the microbes present on the corpse.<ref>{{cite journal|last1=Lehman|first1=Donald C. | name-list-format = vanc |date=April 2014 |title=Forensic Microbiology |journal=Clinical Microbiology Newsletter |volume=36 |issue=7 |pages=49–54 |doi=10.1016/j.clinmicnews.2014.03.001 }}</ref> This would become an integral part of crime solving in later years upon the invention of the microbial clock process.  The microbial timeline in which the body decays has been given the term “microbial clock,” this estimates how long a body has been in a certain place based on microbes present or missing.<ref>{{cite journal | vauthors = Metcalf JL, Wegener Parfrey L, Gonzalez A, Lauber CL, Knights D, Ackermann G, Humphrey GC, Gebert MJ, Van Treuren W, Berg-Lyons D, Keepers K, Guo Y, Bullard J, Fierer N, Carter DO, Knight R | display-authors = 6 | title = A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system | journal = eLife | volume = 2 | pages = e01104 | date = October 2013 | pmid = 24137541 | doi = 10.7554/eLife.01104 }}</ref> The succession of bacterial species populating the body after a period of four days is an indicator of minimum time since death (MTD).<ref>{{cite journal | vauthors = Hauther KA, Cobaugh KL, Jantz LM, Sparer TE, DeBruyn JM | title = Estimating Time Since Death from Postmortem Human Gut Microbial Communities | journal = Journal of Forensic Sciences | volume = 60 | issue = 5 | pages = 1234–40 | date = September 2015 | pmid = 26096156 | doi = 10.1111/1556-4029.12828 }}</ref> The presence or absence of maggots, as well as their age, can also be used to determine time of death; If the maggot is only a few days old, then the cadaver could not have been dead for longer than this time.<ref>{{cite journal | vauthors = Erzinçlioglu Z | title = Forensic entomology | journal = Clinical Medicine | volume = 3 | issue = 1 | pages = 74–6 | date = 1 January 2003 | pmid = 12617420 | doi = 10.7861/clinmedicine.3-1-74 }}</ref>
+==== Microbial forensics ====
-<br />
+As the necrobiome deals with the various communities of bacteria and organisms that catalyze the decomposition of plants and animals (See Figure 1), this particular biome is an increasingly vital part of [[forensic science]]. The microbes occupying the space underneath and around a decomposing body are unique to it--similar to how fingerprints are exclusively unique to only one person.<ref>{{cite journal | vauthors = Franzosa EA, Huang K, Meadow JF, Gevers D, Lemon KP, Bohannan BJ, Huttenhower C | title = Identifying personal microbiomes using metagenomic codes | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 112 | issue = 22 | pages = E2930-8 | date = June 2015 | pmid = 25964341 | doi = 10.1073/pnas.1423854112 }}</ref> Using this differentiation, forensic investigators at a crime scene are able to distinguish between burial sites. This would provide concrete factual information about how long the body has been there and the predicted area in which the death possibly occurred.<ref>{{cite journal | vauthors = Benbow ME, Lewis AJ, Tomberlin JK, Pechal JL | title = Seasonal necrophagous insect community assembly during vertebrate carrion decomposition | journal = Journal of Medical Entomology | volume = 50 | issue = 2 | pages = 440–50 | date = March 2013 | pmid = 23540134 | doi = 10.1603/me12194 }}</ref> As the progression of research regarding microbial forensics and the necrobiome continues to be refined and improved, the need for new forensic scientists and microbiologists becomes increasingly necessary. When a crime such as murder has transpired, a team of crime scene specialists or forensic science experts are called to the scene to collect evidence and examine the body.<ref>{{cite journal|last1=Lutui|first1=Raymond | name-list-format = vanc |date=November 2016|title=A multidisciplinary digital forensic investigation process model|journal=Business Horizons|volume=59|issue=6|pages=593–604|doi=10.1016/j.bushor.2016.08.001}}</ref> These experts range from forensic odontologists to forensic microbiologists (See Figure 2). Together, they can obtain the necessary elements needed in order to properly reconstruct the victim’s demise.
-==== Microbial Forensics ====
-As the necrobiome deals with the various communities of bacteria and organisms that catalyze the decomposition of plants and animals (See Figure 1), this particular biome is an increasingly vital part of [[forensic science]]. The microbes occupying the space underneath and around a decomposing body are unique to it--similar to how fingerprints are exclusively unique to only one person <ref>{{cite journal|last1=Franzosa|first1=Eric A.|last2=Huang|first2=Katherine|last3=Meadow|first3=James F.|last4=Gevers|first4=Dirk|last5=Lemon|first5=Katherine P.|last6=Bohannan|first6=Brendan J. M.|last7=Huttenhower|first7=Curtis|date=2 June 2015|title=Identifying personal microbiomes using metagenomic codes|journal=Proceedings of the National Academy of Sciences|volume=112|issue=22|pages=E2930–E2938|doi=10.1073/pnas.1423854112}}</ref>. Using this differentiation, forensic investigators at a crime scene are able to distinguish between burial sites. This would provide concrete factual information about how long the body has been there and the predicted area in which the death possibly occurred <ref>{{cite journal|last1=Benbow|first1=M. E.|last2=Lewis|first2=A. J.|last3=Tomberlin|first3=J. K.|last4=Pechal|first4=J. L.|date=1 March 2013|title=Seasonal Necrophagous Insect Community Assembly During Vertebrate Carrion Decomposition|journal=Journal of Medical Entomology|volume=50|issue=2|pages=440–450|doi=10.1603/me12194}}</ref>. As the progression of research regarding microbial forensics and the necrobiome continues to be refined and improved, the need for new forensic scientists and microbiologists becomes increasingly necessary. When a crime such as murder has transpired, a team of crime scene specialists or forensic science experts are called to the scene to collect evidence and examine the body <ref>{{cite journal|last1=Lutui|first1=Raymond|date=November 2016|title=A multidisciplinary digital forensic investigation process model|journal=Business Horizons|volume=59|issue=6|pages=593–604|doi=10.1016/j.bushor.2016.08.001}}</ref>. These experts range from forensic odontologists to forensic microbiologists (See Figure 2). Together, they can obtain the necessary elements needed in order to properly reconstruct the victim’s demise.
 === Cadavers and Carcasses ===
-One way many people study how bodies decompose is through the use of [[Body farm|body farms]]. There are seven research facilities in the US that are home to body farms:  University of Tennessee in Knoxville, Western Carolina University, Texas State University, Sam Houston State University, Southern Illinois University, Colorado Mesa University, and University of South Florida. These facilities study the decomposition of cadavers in all possible manners of decay, including in open or frozen environments, buried underground, or within cars <ref>{{cite journal|last1=Wallman|first1=James F.|date=26 October 2017|title=Body farms|journal=Forensic Science, Medicine and Pathology|volume=13|issue=4|pages=487–489|doi=10.1007/s12024-017-9932-z}}</ref>. Through the study of the cadavers, experts examine the microbial timeline and document what’s normal in each stage in the various locations that each body is placed <ref>{{cite journal|last1=Wallman|first1=James F.|date=26 October 2017|title=Body farms|journal=Forensic Science, Medicine and Pathology|volume=13|issue=4|pages=487–489|doi=10.1007/s12024-017-9932-z}}</ref>. An experiment was conducted to study the change in the necrobiome within a carcass and they performed it <ref>Tuccia, Fabiola, Emad Zurgani, Sara Bortolini, and Stefano Vanin. “Experimental Evaluation on the Applicability of Necrobiome Analysis in Forensic Veterinary Science.” MicrobiologyOpen 8, no. 9 (September 2019). https://doi.org/10.1002/mbo3.828</ref>. The experiment was conducted to study the relative abundance of organisms in the necrobiome and the changes that occur during three different stages. For the experiment, they used six dead rabbits purchased from a pet food company. The rabbits were purchased from Kiezebank and exposed on top of a roof at the University of Huddersfield in West Yorkshire, United Kingdom. The rabbits were dead prior to purchase. Three of the rabbits’ fur were removed from the torso to identify any difference in necrobiome abundance. Samples were collected from inside of the mouth, the upper skin of the torso exposed to the air environment, and the bottom skin of the torso that’s touched by the soil. Active, advanced, and decay stages were examined, and proteobacteria were the most abundant present, followed by Firmicutes, Bacteroidetes, and Actinobacteria during the active stage of decomposition (Figure 3).  During the advanced stage of decomposition, Proteobacteria decreased from 99.4% to 81.6% in the oral cavity but were most abundant in the non-fur samples. It was distinguished that Firmicutes were the most abundant for the skin samples in both fur and non-fur samples. Finally, Proteobacteria was most abundant in the soil interface during the beginning of decomposition in both fur and non-fur samples. Also, they noted that Actinobacteria was the least abundant in the active stage and decreased even more during the dry stage <ref>Tuccia, Fabiola, Emad Zurgani, Sara Bortolini, and Stefano Vanin. “Experimental Evaluation on the Applicability of Necrobiome Analysis in Forensic Veterinary Science.” MicrobiologyOpen 8, no. 9 (September 2019). https://doi.org/10.1002/mbo3.828</ref>.
+One way many people study how bodies decompose is through the use of [[Body farm|body farms]]. There are seven research facilities in the US that are home to body farms:  University of Tennessee in Knoxville, Western Carolina University, Texas State University, Sam Houston State University, Southern Illinois University, Colorado Mesa University, and University of South Florida. These facilities study the decomposition of cadavers in all possible manners of decay, including in open or frozen environments, buried underground, or within cars.<ref>{{cite journal | vauthors = Wallman JF | title = Body farms | journal = Forensic Science, Medicine, and Pathology | volume = 13 | issue = 4 | pages = 487–489 | date = December 2017 | pmid = 29075978 | doi = 10.1007/s12024-017-9932-z }}</ref> Through the study of the cadavers, experts examine the microbial timeline and document what’s normal in each stage in the various locations that each body is placed.<ref>{{cite journal | vauthors = Wallman JF | title = Body farms | journal = Forensic Science, Medicine, and Pathology | volume = 13 | issue = 4 | pages = 487–489 | date = December 2017 | pmid = 29075978 | doi = 10.1007/s12024-017-9932-z }}</ref> An experiment was conducted to study the change in the necrobiome within a carcass and they performed it.<ref name="pmid30861327">{{cite journal | vauthors = Tuccia F, Zurgani E, Bortolini S, Vanin S | title = Experimental evaluation on the applicability of necrobiome analysis in forensic veterinary science | journal = MicrobiologyOpen | volume = 8 | issue = 9 | pages = e00828 | date = September 2019 | pmid = 30861327 | pmc = 6741123 | doi = 10.1002/mbo3.828 }}</ref> The experiment was conducted to study the relative abundance of organisms in the necrobiome and the changes that occur during three different stages. For the experiment, they used six dead rabbits purchased from a pet food company. The rabbits were purchased from Kiezebank and exposed on top of a roof at the University of Huddersfield in West Yorkshire, United Kingdom. The rabbits were dead prior to purchase. Three of the rabbits’ fur were removed from the torso to identify any difference in necrobiome abundance. Samples were collected from inside of the mouth, the upper skin of the torso exposed to the air environment, and the bottom skin of the torso that’s touched by the soil. Active, advanced, and decay stages were examined, and proteobacteria were the most abundant present, followed by Firmicutes, Bacteroidetes, and Actinobacteria during the active stage of decomposition (Figure 3).  During the advanced stage of decomposition, Proteobacteria decreased from 99.4% to 81.6% in the oral cavity but were most abundant in the non-fur samples. It was distinguished that Firmicutes were the most abundant for the skin samples in both fur and non-fur samples. Finally, Proteobacteria was most abundant in the soil interface during the beginning of decomposition in both fur and non-fur samples. Also, they noted that Actinobacteria was the least abundant in the active stage and decreased even more during the dry stage.<ref name="pmid30861327" />
 === Decomposition ===
-The manner in which bacteria colonize a cadaver is predictable when examining the time since death<ref name=":7">{{Cite journal|last=Finley|first=Sheree J.|last2=Benbow|first2=M. Eric|last3=Javan|first3=Gulnaz T.|date=2015-05-01|title=Microbial communities associated with human decomposition and their potential use as postmortem clocks|url=https://doi.org/10.1007/s00414-014-1059-0|journal=International Journal of Legal Medicine|language=en|volume=129|issue=3|pages=623–632|doi=10.1007/s00414-014-1059-0|issn=1437-1596}}</ref>. Only recent studies have taken place to determine if bacteria alone can inform the postmortem interval <ref name=":5">{{Citation|last=Hyde|first=Embriette R.|title=Microbial communities associated with decomposing corpses|date=2017|url=https://onlinelibrary.wiley.com/doi/abs/10.1002/9781119062585.ch10|work=Forensic Microbiology|pages=245–273|publisher=John Wiley & Sons, Ltd|language=en|doi=10.1002/9781119062585.ch10|isbn=978-1-119-06258-5|access-date=2020-04-28|last2=Metcalf|first2=Jessica L.|last3=Bucheli|first3=Sibyl R.|last4=Lynne|first4=Aaron M.|last5=Knight|first5=Rob}}</ref>. Bacteria responsible for decomposing cadavers can be difficult to study because the bacteria found on a cadaver varies and changes quickly<ref>Vass, A. (2001) Beyond the grave—understanding human decomposition. Microbiology Today, 28(28), 190–192</ref><ref name=":5" />. Bacteria can be brought to a cadaver by scavengers, air, or water<ref name=":6">{{Cite journal|last=Hyde|first=Embriette R.|last2=Haarmann|first2=Daniel P.|last3=Lynne|first3=Aaron M.|last4=Bucheli|first4=Sibyl R.|last5=Petrosino|first5=Joseph F.|date=2013-10-30|title=The Living Dead: Bacterial Community Structure of a Cadaver at the Onset and End of the Bloat Stage of Decomposition|url=https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077733|journal=PLOS ONE|language=en|volume=8|issue=10|pages=e77733|doi=10.1371/journal.pone.0077733|issn=1932-6203|pmc=PMC3813760|pmid=24204941}}</ref>. Other environmental factors like temperature and soil can impact the microbes found on a cadaver <ref name=":6" />. Fortunately, microbial colonization between humans and animals is so similar, that animal models can be used to understand the decomposition process for humans<ref>{{Cite journal|last=Burcham|first=Z.M.|last2=Hood|first2=J.A.|last3=Pechal|first3=J.L.|last4=Krausz|first4=K.L.|last5=Bose|first5=J.L.|last6=Schmidt|first6=C.J.|last7=Benbow|first7=M.E.|last8=Jordan|first8=H.R.|date=2016-07|title=Fluorescently labeled bacteria provide insight on post-mortem microbial transmigration|url=https://linkinghub.elsevier.com/retrieve/pii/S0379073816301050|journal=Forensic Science International|language=en|volume=264|pages=63–69|doi=10.1016/j.forsciint.2016.03.019}}</ref>. Human cadavers are used for research, but animal models provide larger sample sizes and produce more controlled studies<ref name=":5" /><ref name=":7" />. Swine models have been used repeatedly to understand the human decomposition process in terrestrial environments<ref>{{Cite journal|last=Carter|first=David O.|last2=Metcalf|first2=Jessica L.|last3=Bibat|first3=Alexander|last4=Knight|first4=Rob|date=2015-03-04|title=Seasonal variation of postmortem microbial communities|url=http://dx.doi.org/10.1007/s12024-015-9667-7|journal=Forensic Science, Medicine, and Pathology|volume=11|issue=2|pages=202–207|doi=10.1007/s12024-015-9667-7|issn=1547-769X}}</ref><ref>{{Cite journal|last=Pechal|first=Jennifer L.|last2=Crippen|first2=Tawni L.|last3=Tarone|first3=Aaron M.|last4=Lewis|first4=Andrew J.|last5=Tomberlin|first5=Jeffery K.|last6=Benbow|first6=M. Eric|date=2013-11-12|title=Microbial Community Functional Change during Vertebrate Carrion Decomposition|url=http://dx.doi.org/10.1371/journal.pone.0079035|journal=PLoS ONE|volume=8|issue=11|pages=e79035|doi=10.1371/journal.pone.0079035|issn=1932-6203}}</ref>. Swine are suitable for studying human decomposition because of their size, sparse hairs, and similar bacteria found in their GI tracts<ref>{{Cite journal|last=Kenneth G. Schoenly|last2=Neal H. Haskell|last3=David K. Mills|last4=Carine Bieme-Ndi|last5=Kristle Larsen|last6=Yer Lee|date=2006-09-01|title=Recreating Death's Acre in the School Yard: Using Pig Carcasses as Model Corpses, to Teach Concepts of Forensic Entomology & Ecological Succession|url=http://dx.doi.org/10.2307/4452028|journal=The American Biology Teacher|volume=68|issue=7|pages=402–410|doi=10.2307/4452028|issn=0002-7685}}</ref>.
+The manner in which bacteria colonize a cadaver is predictable when examining the time since death.<ref name=":7">{{cite journal | vauthors = Finley SJ, Benbow ME, Javan GT | title = Microbial communities associated with human decomposition and their potential use as postmortem clocks | journal = International Journal of Legal Medicine | volume = 129 | issue = 3 | pages = 623–32 | date = May 2015 | pmid = 25129823 | doi = 10.1007/s00414-014-1059-0 | url = https://doi.org/10.1007/s00414-014-1059-0 }}</ref> Only recent studies have taken place to determine if bacteria alone can inform the postmortem interval.<ref name=":5">{{cite journal | vauthors = Hyde ER, Metcalf JL, Bucheli SR, Lynne AM, Knight R |title=Microbial communities associated with decomposing corpses|date=2017 |work=Forensic Microbiology |pages=245–273 |publisher=John Wiley & Sons, Ltd |doi= 10.1002/9781119062585.ch10 |isbn= 978-1-119-06258-5 }}</ref> Bacteria responsible for decomposing cadavers can be difficult to study because the bacteria found on a cadaver varies and changes quickly.<ref>{{cite journal | vauthors = Vass A | date = 2001 | title = Beyond the grave—understanding human decomposition. | journal = Microbiology Today | volume = 28 | issue = 28 | pages = 190–192 }}</ref><ref name=":5" /> Bacteria can be brought to a cadaver by scavengers, air, or water.<ref name=":6">{{cite journal | vauthors = Hyde ER, Haarmann DP, Lynne AM, Bucheli SR, Petrosino JF | title = The living dead: bacterial community structure of a cadaver at the onset and end of the bloat stage of decomposition | journal = PloS One | volume = 8 | issue = 10 | pages = e77733 | date = 2013-10-30 | pmid = 24204941 | pmc = 3813760 | doi = 10.1371/journal.pone.0077733 }}</ref> Other environmental factors like temperature and soil can impact the microbes found on a cadaver.<ref name=":6" /> Fortunately, microbial colonization between humans and animals is so similar, that animal models can be used to understand the decomposition process for humans.<ref>{{cite journal | vauthors = Burcham ZM, Hood JA, Pechal JL, Krausz KL, Bose JL, Schmidt CJ, Benbow ME, Jordan HR | display-authors = 6 | title = Fluorescently labeled bacteria provide insight on post-mortem microbial transmigration | journal = Forensic Science International | volume = 264 | pages = 63–9 | date = July 2016 | pmid = 27032615 | doi = 10.1016/j.forsciint.2016.03.019 }}</ref> Human cadavers are used for research, but animal models provide larger sample sizes and produce more controlled studies.<ref name=":5" /><ref name=":7" /> Swine models have been used repeatedly to understand the human decomposition process in terrestrial environments.<ref>{{cite journal | vauthors = Carter DO, Metcalf JL, Bibat A, Knight R | title = Seasonal variation of postmortem microbial communities | journal = Forensic Science, Medicine, and Pathology | volume = 11 | issue = 2 | pages = 202–7 | date = June 2015 | pmid = 25737335 | doi = 10.1007/s12024-015-9667-7 | url = http://dx.doi.org/10.1007/s12024-015-9667-7 }}</ref><ref>{{cite journal | vauthors = Pechal JL, Crippen TL, Tarone AM, Lewis AJ, Tomberlin JK, Benbow ME | title = Microbial community functional change during vertebrate carrion decomposition | journal = PloS One | volume = 8 | issue = 11 | pages = e79035 | date = 2013-11-12 | pmid = 24265741 | doi = 10.1371/journal.pone.0079035 }}</ref> Swine are suitable for studying human decomposition because of their size, sparse hairs, and similar bacteria found in their GI tracts.<ref>{{cite journal | vauthors = Schoenly KG, Haskell NH, Mills DK, Bieme-Ndi C, Larsen K, Lee Y |date=2006-09-01|title=Recreating Death's Acre in the School Yard: Using Pig Carcasses as Model Corpses, to Teach Concepts of Forensic Entomology & Ecological Succession |journal=The American Biology Teacher |volume=68 |issue=7 |pages=402–410 |doi=10.2307/4452028 }}</ref>
 === Technology and techniques ===
-An algorithm has been developed to accurately predict time since death with an accuracy of within two days <ref>Johnson, Hunter R., Donovan D. Trinidad, Stephania Guzman, Zenab Khan, James V. Parziale, Jennifer M. DeBruyn, and Nathan H. Lents. “A Machine Learning Approach for Using the Postmortem Skin Microbiome to Estimate the Postmortem Interval.” Edited by Raymond Schuch. PLOS ONE 11, no. 12 (December 22, 2016): e0167370. https://doi.org/10.1371/journal.pone.0167370.</ref>. This multi-step process focuses on criminal investigations and forensics science to obtain the post-mortem interval. Before the continuous development, there was no universal algorithm. Algorithms are complicated due to various factors in both biotic and abiotic which leads to a bimolecular process<ref name=":2" />. One change can make a dramatic difference to the necrobiome (i.e. body mass index, the clothing on the carcass, body temperature, contact with the ground).
+An algorithm has been developed to accurately predict time since death with an accuracy of within two days.<ref name="pmid28005908">{{cite journal | vauthors = Johnson HR, Trinidad DD, Guzman S, Khan Z, Parziale JV, DeBruyn JM, Lents NH | title = A Machine Learning Approach for Using the Postmortem Skin Microbiome to Estimate the Postmortem Interval | journal = Plos One | volume = 11 | issue = 12 | pages = e0167370 | date = 2016 | pmid = 28005908 | pmc = 5179130 | doi = 10.1371/journal.pone.0167370 }}</ref> This multi-step process focuses on criminal investigations and forensics science to obtain the post-mortem interval. Before the continuous development, there was no universal algorithm. Algorithms are complicated due to various factors in both biotic and abiotic which leads to a bimolecular process.<ref name=":2" /> One change can make a dramatic difference to the necrobiome (i.e. body mass index, the clothing on the carcass, body temperature, contact with the ground).
-It is difficult to come to an agreement on technology and technique because of different rules for each region. They suggested that because of genetic variation, DNA profiling is the best approach; however, algorithms developed within the United States are not applicable to other regions due to environmental variations. Currently, the majority of algorithms use genomic analysis for understanding microbial species that inhabit soil and their contribution to the decomposition process<ref name=":2" />.Techniques for analyzing the necrobiome have now been coupled with forensic entomology, such as phospholipid fatty acid (PLFA) analysis <ref>Parkinson, Rachel A., Kerith-Rae Dias, Jacqui Horswell, Paul Greenwood, Natasha Banning, Mark Tibbett, and Arpad A. Vass. “Microbial Community Analysis of Human Decomposition on Soil.” In Criminal and Environmental Soil Forensics, edited by Karl Ritz, Lorna Dawson, and David Miller, 379–94. Dordrecht: Springer Netherlands, 2009. https://doi.org/10.1007/978-1-4020-9204-6_24</ref>, total soil fatty acid methyl esters <ref>Parkinson, Rachel A., Kerith-Rae Dias, Jacqui Horswell, Paul Greenwood, Natasha Banning, Mark Tibbett, and Arpad A. Vass. “Microbial Community Analysis of Human Decomposition on Soil.” In Criminal and Environmental Soil Forensics, edited by Karl Ritz, Lorna Dawson, and David Miller, 379–94. Dordrecht: Springer Netherlands, 2009. https://doi.org/10.1007/978-1-4020-9204-6_24.</ref>, and DNA profiling <ref>Parkinson, Rachel A., Kerith-Rae Dias, Jacqui Horswell, Paul Greenwood, Natasha Banning, Mark Tibbett, and Arpad A. Vass. “Microbial Community Analysis of Human Decomposition on Soil.” In Criminal and Environmental Soil Forensics, edited by Karl Ritz, Lorna Dawson, and David Miller, 379–94. Dordrecht: Springer Netherlands, 2009. https://doi.org/10.1007/978-1-4020-9204-6_24.</ref>. Pig carcasses have also become a tool to understand human microbiology, minimizing the issue of variation that exists when using human cadavers as study subjects <ref>Parkinson, Rachel A., Kerith-Rae Dias, Jacqui Horswell, Paul Greenwood, Natasha Banning, Mark Tibbett, and Arpad A. Vass. “Microbial Community Analysis of Human Decomposition on Soil.” In Criminal and Environmental Soil Forensics, edited by Karl Ritz, Lorna Dawson, and David Miller, 379–94. Dordrecht: Springer Netherlands, 2009. https://doi.org/10.1007/978-1-4020-9204-6_24.</ref>. This technology is used to simplify the sample collection into sequences that scientists can read. The simplified sequence of the necrobiome is run through a data bank to match the name of it. Due to the lack of universal algorithm technology, there is a knowledge gap in various platforms across different regions of the world. In order to close that gap, there needs to be an expansion of the technology. However, there are a few obstacles, including identifying needs, research, prototype development, acceptance, and adoption <ref>Metcalf, Jessica L. “Estimating the Postmortem Interval Using Microbes: Knowledge Gaps and a Path to Technology Adoption.” Forensic Science International: Genetics 38 (January 2019): 211–18. https://doi.org/10.1016/j.fsigen.2018.11.004</ref>. Overcoming these obstacles would assist many organizations that are involved with forensic science. Also, it would increase the understanding of the necrobiome and growth of developing a successful accurate multi-step experiment. The samples are loaded into a machine to generate and analyze DNA sequences of the microbiome. Algorithms are done in a lab on a computer program to read and match the sequences within the data bank. The results return very quickly within a few minutes to the latest days.
+It is difficult to come to an agreement on technology and technique because of different rules for each region. They suggested that because of genetic variation, DNA profiling is the best approach; however, algorithms developed within the United States are not applicable to other regions due to environmental variations. Currently, the majority of algorithms use genomic analysis for understanding microbial species that inhabit soil and their contribution to the decomposition process.<ref name=":2" />Techniques for analyzing the necrobiome have now been coupled with forensic entomology, such as phospholipid fatty acid (PLFA) analysis,<ref name = "Parkinson_2009">{{cite book | vauthors = Parkinson RA, Dias KR, Horswell J, Greenwood P, Banning N, Tibbett M, Vass AA | chapter =  Microbial community analysis of human decomposition on soil. | title = Criminal and environmental soil forensics | date = 2009 | pages = 379–394 | publisher = Springer | location = Dordrecht | doi = 10.1007/978-1-4020-9204-6_24 }} </ref> total soil fatty acid methyl esters,<ref name = "Parkinson_2009" /> and DNA profiling.<ref name = "Parkinson_2009" /> Pig carcasses have also become a tool to understand human microbiology, minimizing the issue of variation that exists when using human cadavers as study subjects.<ref name = "Parkinson_2009" /> This technology is used to simplify the sample collection into sequences that scientists can read. The simplified sequence of the necrobiome is run through a data bank to match the name of it. Due to the lack of universal algorithm technology, there is a knowledge gap in various platforms across different regions of the world. In order to close that gap, there needs to be an expansion of the technology. However, there are a few obstacles, including identifying needs, research, prototype development, acceptance, and adoption.<ref name="pmid30448529">{{cite journal | vauthors = Metcalf JL | title = Estimating the postmortem interval using microbes: Knowledge gaps and a path to technology adoption | journal = Forensic Science International. Genetics | volume = 38 | issue = | pages = 211–218 | date = January 2019 | pmid = 30448529 | doi = 10.1016/j.fsigen.2018.11.004 }}</ref> Overcoming these obstacles would assist many organizations that are involved with forensic science. Also, it would increase the understanding of the necrobiome and growth of developing a successful accurate multi-step experiment. The samples are loaded into a machine to generate and analyze DNA sequences of the microbiome. Algorithms are done in a lab on a computer program to read and match the sequences within the data bank. The results return very quickly within a few minutes to the latest days.
 === Conclusion ===
@@ Line 34: / Line 32: @@
 == References ==
-{{reflist|30em}}
+{{reflist}}
 {{DEFAULTSORT:Necrobiome}}