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New Approaches in Estimating the Post-Mortem Interval (PMI) – the Human Microbiome As An New approaches in estimating the post-mortem interval (PMI) – The human microbiome as an indicator of the time since death Magdalena Birkl 11406739 Master Forensic Science Supervisor: Prof. Dr. Roelof-Jan Oostra Co-assessor: Dr. Annemieke van Dam Date: 24.2.2018 Table of Contents List of abbreviations ........................................................................................................................................... 3 Abstract .............................................................................................................................................................. 4 1. Introduction and background .................................................................................................................... 5 1.1. Research question ......................................................................................................................... 5 1.2. Background ................................................................................................................................... 5 2. Overview studied literature ....................................................................................................................... 7 2.1. Human cadaver studies ................................................................................................................. 7 2.1.1. Sample size ....................................................................................................................... 7 2.1.2. Sampling points ................................................................................................................ 7 2.1.3. Sampling region ............................................................................................................... 7 2.1.4. Sampling technique.......................................................................................................... 8 2.1.5. Analysis method ............................................................................................................... 9 2.1.6. Results and conclusions ................................................................................................... 9 2.2. Non-human cadaver studies ....................................................................................................... 11 2.2.1. Animal model and sample size....................................................................................... 12 2.2.2. Test condition ................................................................................................................ 12 2.2.3. Sampling points .............................................................................................................. 12 2.2.4. Sampling region ............................................................................................................. 13 2.2.5. Sampling technique........................................................................................................ 13 2.2.6. Analysis method ............................................................................................................. 14 2.2.7. Results and conclusions ................................................................................................. 14 2.3. Summary ..................................................................................................................................... 16 3. Discussion of literature ............................................................................................................................ 19 3.1. Differences between human cadaver and animal models ......................................................... 19 3.2. Influence of extrinsic and intrinsic factors .................................................................................. 19 3.3. Influence of the sampling strategy ............................................................................................. 19 3.4. Additional possible influencing factors which raise questions ................................................... 19 3.5. The shift from aerobic to anaerobic taxa .................................................................................... 20 3.6. Influence of temperature on seasonal and annual variation ..................................................... 20 3.7. Different decomposition patterns .............................................................................................. 20 3.8. Influence of insects and soil bacteria .......................................................................................... 20 3.9. Influence of the used methodology ............................................................................................ 21 4. Conclusion and recommendations .......................................................................................................... 22 5. References ............................................................................................................................................... 23 Appendix .......................................................................................................................................................... 25 2 Magdalena Birkl, 11406739 List of abbreviations ADD Accumulated degree days C.perfringens Clostridium perfringens CDH Cumulative degree hours DNA Deoxyribonucleic acid MALDI-TOF Matrix-assisted laser desorption/ionisation time of flight mass spectrometry MLN Mesenteric lymph node NGS Next generation sequencing PMI Post-mortem interval qPCR Quantitative polymerase chain reaction rRNA Ribosomal ribonucleic acid RT-PCR Reverse transcription polymerase chain reaction S.aureus Staphylococcus aureus T-RFLP Terminal restriction fragment length polymorphism 3 Magdalena Birkl, 11406739 Abstract Abstract The determination of the Post-Mortem Interval (PMI) is a crucial step in a death investigation. Several currently used methods however are prone to errors and result in large uncertainties in time since death. As microbes are ubiquitous in nature and as the human microbiome is well studied in the living human it is considered to play a major role in the human decomposition process. A few studies have been conducted up to now to investigate the microbial migration throughout the decomposition process with means of animal models, such as pig and mice, and human cadavers. The investigation of external and internal sampling sites during the decomposition process revealed a distinctive shift from aerobic to anaerobic bacterial communities. However, also several factors which could influence the accuracy of the proposed method were identified, for example inter- individual and sex-specific variations as well as seasonal variations. Nonetheless, 4 study groups were able to establish a prediction model for the estimation of the post-mortem interval (PMI). Due to the small sample size and many limiting factors, additional research is recommended. Keywords: microbiome, PMI, human decomposition, human cadavers, animal cadavers 4 Magdalena Birkl, 11406739 1. Introduction and background 1. Introduction and background 1.1. Research question The main focus of this literature thesis lies on discussing the current status in the field of post- mortem interval (PMI) estimation using the human microbiome. Questions that arise in this context are for example to which extent the human microbiome is involved in the decomposition process itself. In addition, it has to be evaluated if a method which uses the microbiome adds value to the currently used techniques and is able to estimate the PMI more accurate. 1.2. Background The determination of the PMI is a crucial step in every death investigation since it helps identifying suspects and the confirmation and rejection of alibis (1). Therefore, a reliable and accurate method is necessary to assess the PMI and to use the respective results as physical evidence before court. Currently several standard methods are t used for the PMI estimation having all specific limitations These limitations are the trigger for studying a new approach for the PMI estimation. The time since death can be evaluated by various methods which focus on different aspects of the decomposition process. One well known method is based on findings in the field of entomology (2). With help of insects and the deposition of their eggs, the PMI can be established. Each insect species is differently attracted by the cadaver and will place their eggs at specific points in time depending on the decomposition status. The development of the insect larvae is extensively studied and can be used as a time indicator. However, placement of insect eggs is dependent on the accessibility of the cadaver and on insect activity, being dependent on temperature, weather conditions, seasonal variations and geographic location (3). This leads to possible inaccuracies if the access to the body is not possible or the egg deposition is delayed due to reduced insect activity, for example in the winter season (4). Another often used method is the mortis triad which consists of algor mortis, livor mortis and rigor mortis (2). This method focuses on the settling of the blood in the lower parts of the body (livor mortis), the stiffening of the body post-mortem (rigor mortis) and the decrease of the body temperature after death (algor mortis). All 3 tests are prone to inter-individual variation and are in the case of livor and rigor mortis only of a temporal duration. For the estimation of the PMI with the means of the body cooling (algor mortis), Henßge developed
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