Using high-throughput sequencing of the thanatomicrobiome of criminal cases Gulnaz T Javan, Ph.D. Alabama State University, Department of Physical Sciences,

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Presentation transcript:

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 2007 Committing $140 million Coevolution of human microbiome 90% of the cells in human body are microbe

HMP 10 trillion human cells 20,000 human genes 100 trillion microbial cells 20,000 human genes 2-20 million microbial genes human human

Dr. Javan and her team ASU, Montgomery, AL Dr. Sari Tuomisto Tampere, Finland

ASU researchers hope to help solve homicides 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-microbes http://www.montgomeryadvertiser.com/story/news/local/2014/07/08/asu-researchers-hope-help-solve-homicides/12327763/

What 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) Extraction Organs: Brain, Heart, Liver and Spleen Bead-beater Centrifuge

Method-I : Phenol-Chloroform Extraction Organs: Brain, Heart, Liver and Spleen Bead-beater Centrifuge

Method-II: Q-tip Organs: Brain, Heart, Liver and Spleen

Phylogenetic tree of life & 16S ribosomal RNA structure Universal primers 27F and 1492R Carl Woese - 1977

Amplifications

Case 1: Microbial Composition Sampling method PC extraction Same two dominant spp Case 1: Microbial Composition

Case 1: Liver vs Other Organs Liver showed higher diversity Case 1: Liver vs Other Organs

Microbial composition in the liver PC extraction comparison Case 1 has higher diversity in liver Microbial composition in the 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 Process Fresh Stage Minimal disturbance of the soil Intrinsic bacteria and parasites begin the internal decomposition I. Bloat Gases produced by the enteric microorganisms cause bloat Can detect the odor of decaying flesh Maggot activity begins II. Active Decay Tissues distend & cadaveric fluids efflux into the soil from orifices (nose, mouth, ears, genital area & rectum) III. Advanced Decay Maggot activity increases during this stage Maggot larvae migrate into the underlying grave-soil IV. Dry Remains Gradual decease in cadaveric nutrient concentrations

Future Studies: Microbial Diversity Fresh Stage Endogenous enteric-associated bacteria Bloat Aerobic microorganisms predominate, depleting the cadaver of O2 Then endogenous anaerobes, Firmicutes in the Lactobacillaceae and Bacteroidetes in the Bacteroidaceae families, increase Active Decay Endogenous 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 flourish And facultative anaerobic Gammaproteobacteria, particularly of the Enterobacteriaceae family to flourish Dry Remains Aerobic bacteria and fungi remain active for months

Q tip More microbial diversity No toxic chemicals e.g.phenol…. Less time Easy 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 Same 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

Conclusion Q tip method provides higher microbial diversity than phenol-chloroform method Liver sample cases showed different microbial diversity Goal is to identify a unique microbe that can help with PMI Collection of samples and study are ongoing….

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

NSF HRD 1401075 Acknowledgements I thank Stephen Boudreau, M.D. and the Alabama Department of Forensic Sciences Montgomery Medical Laboratory for providing our case samples. NSF HRD 1401075 Ismail Can, M.Sc. student

Questions