A Comparative Study Of Terrestrial Cadaveric Bacterial Communities Succession Patterns And PMI Inference In Two Environments

Objective:Postmortem interval(PMI)inference is an important problem in forensic medicine,which is important for the detection of various judicial cases.The use of microbiomics for PMI inference has become a hot research topic at this stage.Although a large number of studies have been reported on the application of microbiomics for PMI inference of terrestrial carcasses,most of them are focused on exposed air carcasses,and fewer studies are related to buried carcasses,and the studies only explore the carcasses bacterial community succession pattern and PMI model construction in a single environment.The similarities and differences in the succession patterns of carcasses bacterial community and PMI model construction of terrestrial carcasses under two environments(exposed air and buried conditions)have not been discussed in the study.In this study,rat carcasses under exposed air and burial conditions were used as the study subjects to detect microbial communities during decay using 16S r RNA sequencing technology,and to compare the evolution patterns of bacterial communities of microbial communities in the heart,muscle,cecum and soil beneath buried carcasses during decay in the two environments,as well as the similarities and differences of the constructed PMI inference models.In order to provide a theoretical basis for PMI inference using microbiomics methods for terrestrial carcasses.Methods:1.Sample collection:75 9-week-old male healthy Sprague-Dewley rats,weighing 250～300 g,were selected and executed by the cervical dislocation method,and the rat carcasses were divided into two groups:one group was the experimental group,which was buried in soil at a depth of 1 m after death;the other group was the control group,which was stored in a constant temperature and humidity chamber at 25°C and 50%relative humidity after death.At 0 d,1 d,3 d,5 d,7 d,10 d,15 d and 30 d after death(where 0 d was the common time point).Destructive sampling of the carcasses was performed,and the heart,muscle,cecum and soil beneath the carcasses of the experimental group were collected from the rats(n=5).Samples were collected at each time point and placed in liquid nitrogen for 1 h after fixation,and then stored in a-80°C refrigerator.2.After collecting the above samples,DNA was extracted from each sample and sequenced using 16S r RNA amplicons to detect and obtain information on the bacterial community of each sampling site.3.Data analysis:The obtained data were analyzed by data quality assessment,sequence splicing,chimera removal and short sequence analysis,ASV statistical analysis and annotation,species abundance statistics and community composition analysis,Alpha diversity analysis and Beta diversity analysis between samples.To obtain the microbial community succession patterns of each group of rat carcasses and compare the similarities and differences.A random forest(RF)algorithm was used to construct PMI prediction models for each of the two environments and compare the similarities and differences.Results:1.The corpses of the buried group entered the active stage of decay at day 3 of the experiment,while the control corpses entered the active stage of decay at day 5,and the corpses of both groups entered the late stage of decay at day 30.The dominant bacteria of each sample and its changes were different in different environments within each phase.2.Initial sterile organs(heart and muscle samples)showed similar changes in bacterial Alpha diversity in both environments,while initial sterile organs(cecum samples)showed different changes in bacterial Alpha diversity.Environmental factors had an effect on their microbial succession,especially in the initial germinated organisms.The composition of the sterile organism bacterial community was similar in the same environment;the composition of the sterile organism bacterial community was similar in different environments.3.In the same sampling site(cecum samples)in different environments,there is some similarity in the composition of the bacterial community that changes with time of death,that is,there is a core bacterial communities.However,the change pattern of bacterial community succession was not the same.4.PMI inference models based on burial soil samples and cecum samples in both environments were successfully constructed.The PMI inference model constructed for the cecum sample in the burial group had an R~2of 0.93 and a MAE value of 2.41(days)for the test set.The PMI inference model constructed for the buried group gravesite samples had an R~2 of 0.97 and a MAE value of 2.28(days)for the test set.The PMI inference model constructed for the cecum samples of the control group had an R~2 of 0.91 and a MAE value of 4.53(days)for the test set.Species used in the inferential model for both cecum groups were found to have duplication at the genus level for 12 species in both samples.These species have the potential to infer the time to death in different environments.Conclusions:1.The microbial succession pattern of carcasses under deep burial conditions was different from that under exposed air conditions.2.PMI inference models were successfully constructed for two terrestrial environments for grave soil samples and cecum samples.3.In the comparison of the models constructed for cecum samples in different environments,core bacterial communities with potential to infer PMI in different terrestrial environments were identified.This provides a theoretical basis for PMI inference in terrestrial carcasses using microbiomic approaches.