Life After Human Death: The Thanatomicrobiome

Using high-throughput sequencing of the thanatomicrobiome of criminal cases Gulnaz T Javan, Ph.D. Alabama State University, Department of Physical Sciences, Forensic Science Program Montgomery, AL 36104

Human Microbiome Project (HMP)First phase (2007-2012) & Second phase (2013-2015)NIH began the HMP in 2007Committing $140 millionCoevolution of human microbiome90% of the cells in human body are microbe

HMP

10 trillion human cells100 trillion microbial cells

humanhuman20,000 human genes2-20 million microbial genes

Dr. Javan and her teamASU, Montgomery, ALDr. Sari TuomistoTampere, Finland

The National Science Foundation recently awarded the team a $200,000 research grant to further their study. It's the first grant for ASU's forensic science program and will allow 8 undergraduate and 2 graduate students to be involved in molecular biology research (2 years)

http://www.forensicmag.com/articles/2014/09/dirty-world-body-farm-microbeshttp://www.montgomeryadvertiser.com/story/news/local/2014/07/08/asu-researchers-hope-help-solve-homicides/12327763/

ASU researchers hope to help solve homicidesWhat is the Thanatomicrobiome?Thanatos: in Greek Mythology death

Microbe: single-cell living organism, such as bacterium, fungus, protozoan, or virus

Microbiome: collectively all the microbes in the human body; a community of microbes

Subjects

Method-I : Phenol-Chloroform (PC) Extraction10Organs:Brain, Heart, Liver and SpleenBead-beater

Centrifuge

10Method-I : Phenol-Chloroform Extraction11Organs:Brain, Heart, Liver and SpleenBead-beater

Centrifuge

11Method-II: Q-tip12Organs:Brain, Heart, Liver and Spleen

12Phylogenetic tree of life &16S ribosomal RNA structure

Universal primers27F and 1492R

Carl Woese - 1977Amplifications

Case 1: Microbial Composition Sampling method PC extractionSame two dominant spp

Case 1: Liver vs Other OrgansLiver showed higher diversity

Microbial composition in the liverPC extraction comparison Case 1 has higher diversity in liver

Figure 3. Microbial diversity of liver compared to brain, heart, and spleen. According to a thanatomicrobiome study of corpses at various times of death, the liver had higher microbial diversity than the brain, heart, and spleen.

Liver: Why so Diverse?Life style?Last meal?Last medicine?Cause of death?PMI?

Cadaver Decomposition Process22Fresh StageMinimal disturbance of the soilIntrinsic bacteria and parasites begin the internal decomposition

I. BloatGases produced by the enteric microorganisms cause bloatCan detect the odor of decaying fleshMaggot activity begins

IV. Dry RemainsGradual decease in cadaveric nutrient concentrations

II. Active DecayTissues distend & cadaveric fluids efflux into the soil from orifices (nose, mouth, ears, genital area & rectum)

III. Advanced Decay Maggot activity increases during this stageMaggot larvae migrate into the underlying grave-soil

22

Future Studies: Microbial Diversity 23Fresh StageEndogenous enteric-associated bacteria BloatAerobic microorganisms predominate, depleting the cadaver of O2Then endogenous anaerobes, Firmicutes in the Lactobacillaceae and Bacteroidetes in the Bacteroidaceae families, increaseActive DecayEndogenous bacteria from the GI tract and other microorganisms that spread via the blood and lymphatic systems Advanced Decay Exposure of the abdominal cavity to O2 allows aerobic Alphaproteobacteria, Rhizobiales in the Phyllobacteriaceae, Hyphomicrobiaceae and Brucellaceae families to flourishAnd facultative anaerobic Gammaproteobacteria, particularly of the Enterobacteriaceae family to flourish Dry RemainsAerobic bacteria and fungi remain active for months23Q tipMore microbial diversityNo toxic chemicals e.g.phenol.Less timeEasy to do, no expertise required

Case 1: Microbial Composition

Case 1: Higher Microbial Diversity in Liver

Microbial composition in the human brain

Microbial composition in the human spleen

28Same physical/chemical/beading methods

Microbial composition in the human heart

Microbial composition in the human liver

Method comparison: Spleen

Method comparison: Heart

Method comparison: Liver

ConclusionQ tip method provides higher microbial diversity than phenol-chloroform methodLiver sample cases showed different microbial diversityGoal is to identify a unique microbe that can help with PMICollection of samples and study are ongoing.

Future PerspectivesDifferent OTU (Operational Taxonomic Unit) level?Microbial composition and cause of death?Microbial composition and PMI?

AcknowledgementsI thank Stephen Boudreau, M.D. and the Alabama Department of Forensic Sciences Montgomery Medical Laboratory for providing our case samples.

Ismail Can, M.Sc. student

NSF HRD 1401075Questions