3.1 The relative abundance of gut flora in different groups
A total of 7,029,815 raw data and 6,674,323 clean data were obtained by high throughput sequencing, with an effective rate of 94.97% (the ratio of Clean Data to Raw Data). A total of 22,625 OTUs were based on 97% similarity allocated, with an average of 257 OTUs per sample. The total usable sequences were classified into 33 phyla, 49 classes, 108 orders, 203 families, 465 genera and 306 species. Species accumulation boxplot and rarefaction curves of all samples became smooth with the quantity of sequences enlarged, demonstrating that this sequencing profundity could mirror the whole bacterial species richness (Figure. 1). A Venn diagram was applied for comparing the similarities and variances among the communities in the different groups. The eleven groups showed communities of 36 OTUs in shared, with the unique OTUs composed of 84.94%, 90.00%, 74.83%, 87.84%, 21.74%, 14.29%, 5.26%, 10.00%, 16.28%, 23.4%, 26.53%in time points of alive, 0 hour, 8 and 16 hours, day 1, 3, 5, 7, 9, 13, 15, respectively (Figure. 2). Ternary plots displayed by Ternary plot order of VCD package in R software. Figure. 3A indicated that the abundance of Corynebacterium_amycolatum was the highest and the most high-load in h0 among the three time points, and that of Baterium_mpn_isolate_group_2 and Falsiporphyromonas_endometrii were higher in alive, 8 hours after death, respectively. Figure. 3B showed that the richness of Enterococcus_faecalis was increased in 16 hours and day 1, 3 post-mortem, followed by Proteus_mirabilis, which was higher in day 3 post-mortem as compared to the other two time points; Figure. 3C showed that the relative abundance of Enterococcus_faecalis was increased in day 5 post-mortem, and possessed almost same abundance in day 7, 9 post-mortem; Figure. 3D showed Proteus_mirabilis was the highest one among the three time points of day 15 after death and followed by Vagococcus_lutrae, and Enterococcus_faecalis was much higher in day 9, 13 post-mortem than day 15 after death.
3.2 Microbial analysis at different levels
The microbial community structure succession was identified during decomposition, all of the 16S rRNA sequences were classified at phylum, genus and species level. The notable tendencies and fluctuations exhibited in the relative richness of the diverse bacterial taxa in the rectum of the rat cadavers through the decaying process (Figure. 4A, B, C; Figure. 5A, B, C; Table 1). Figure. 4A, B, C showed variations of bacteria with their proportions at different levels and Figure. 5A, B, C showed the relative abundance of 10 topmost bacteria in the study samples.
At the phyla level, Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria existed in wholly time points. Bacteroidetes (54.57%), Firmicutes (45.83%) and Proteobacteria were the dominant phylum in alive samples, 0-hour post-mortem and other time points, respectively. The Bacteroidetes began to show a downward trend and even disappeared, rising to 5.52% in 15 days post-mortem samples. The relative abundance of Proteobacteria in alive samples was much lower than 8-hours, day 1, 9 post-mortem, while Bacteroidetes was greatly higher in alive samples than 8-hours, day 3, 5, 7, 9, 13, 15, and Actinobacteria was significantly higher in 0-hour, day 5, 7, 9, 13, 15 post-mortem (P<0.05).
At the genus level, Lactobacillus, Enterococcus were appeared as the dominant genera before day 1 post-mortem and day 3-13, respectively, and Helicobacter disappeared on 7, 9, 15 post-mortem intervals and Proteus was the most abundant in day 15 post-mortem. The relative abundance of rectum flora in alive samples was greatly lower than in day 3, 5, 7, 9, 13 post-mortem intervals, nevertheless, Helicobacter was much higher in these days (P<0.05). The proportion of Proteus and Lactobacillus showed significantly lower in alive and 0-hour than in day 13 post-mortem samples, while vagococcus displayed opposite result (P<0.05).
At the species level, the 10 topmost species existed in 8 hours post-mortem, but Clostridium_sporogenes and Falsiporphyromonas_endometrii disappeared before day 1 post-mortem and after day 3 post-mortem, respectively. Enterococcus_faecalis and Proteus_mirabilis appeared at the whole decomposition process of 15 days after death, the former one showed a downward trend from the day 5 post-mortem, while the latter one showed an upward trend. Bacterium_mpn-isolate_group_2 disappeared (day 5-13 post-mortem) or decreased after alive. The relative abundance of Enterococcus_faecalis was much lower in alive samples than in day 5, 7, 9, 13, 13, 15 after death, while Bacterium_mpn-isolate_group_2 was distinctly higher in these days (P<0.05). The relative abundance of Corynebacterium_amycolatum and Lactobacillus_intestinalis were greatly higher in 0-hour, day 7, 9, 13, 15 post-mortem intervals, and Lactobacillus_reuteri was higher in alive, 0-hour, 16-hours and day 1 than in day 13 post-mortem.
3.3 Characterization of bacterial diversity and community structure
The whole rectum flora community was evaluated by diversity and richness as calculated at the level of 97% similarity. Alpha diversity indexes of the observed_species, abundance based coverage estimators (ACE) and chao1 values for the rectal bacteria in the alive samples were significantly higher than those in day 5, 7, 9, 13, 15 post-mortem, suggesting that richness and diversity of the rectum flora decline a lot after day 5 post-mortem (Table 2). All of the Alpha diversity indexes were showed in Table 2 that there were having significant differences in overall rectum bacterial community structure in the eleven post-mortem intervals.
The similarity of the gut flora communities of rats in eleven groups were measured by beta diversity metrics such as Non-metric dimensional scaling (NMDS) and Beta diversity heatmap to define the differences of each individual animal gut microbial structure at different post-mortem interval. As displayed in Figure. 6A, the difference coefficients among other groups were almost greater than 0.5, telling that bacteria community in different groups had great diversity. All of the samples were gathered into 11 prime clusters. According to Non-Metric Multi-Dimensional Scaling (NMDS, stress=0.152), the bacterial communities of the gut samples were separated into three clusters between late and early PMI (Figure. 6B). Conspicuously, 8 hours post-mortem could be significantly separated from other groups, but samples of 8 hours post-mortem were clustered, indicating that the gut flora at 8 hours after death differed with other two clusters obviously.
LDA Effect Size (LEfSe) is a biomarker detection and descriptive device for high-dimensional statistics. The LEfSe analysis was performed for comparing the projected bacterial community among 11 time points at different levels (Figure. 6C). The results suggested that the provision of related taxa significantly diverse among overall groups. The LDA scores showed that the relative abundances of Corynebacterium_amycolatum, Entero_isolate_group_2, Bacteroides_uniformis, Enterococcus_faecalis, Streptococcus_gallolyticus_subsp_macedonics and Clostridium_sporogenes were more abundant in 0-h, day 1, 3, 5, 7, 13 post-mortem intervals, respectively, while Proteus_mirabilis ands_Vagococcus_lutrae were in day 15 post-mortem.
The shifts in the probable functions of the gut flora of rats before and after death were inspected by predicting the 16S rRNA genes using PICRUSt (Figure. 7A, B). The top four different pathways at Kyoto Encyclopedia of Genes and Genomes (KEGG) at level 1 were Metabolism, Genetic_Information_Processing, Environmental_Information_Processing and Cellular_Processes and at level 2 were Membrane_Transport, Carbohydrate_Metabolism, Amino_Acid_Metabolism and Replication_and_Repair. The functional pathway related with Environmental_Information_Processing such as Membrane_Transport as the dominant one in the whole time points, then the followed pathways associated with Genetic_Information_ Processing and Metabolism, including Carbohydrate_Metabolism, Amino_Acid_Metabolism and Replication_and_Repair, respectively, possessing large number associated genes in all samples (Figure. S1). The pathways of Environmental_Information_Processing and Organismal_ Systems were significantly higher in rectum bacterial community day 5, 7, 9, 13, 15 after death as compared to alive and Membrane_Transport associated with the first mentioned pathway displaying identical result (Table S1, S2). Amino_Acid_Metabolism, Energy_Metabolism and Metabolism_of_Cofactors_and_Vitamins exhibited significant difference between alive and day 5, 7, 9 post-mortem intervals, while Energy_Metabolism showed notable differences between alive and after day 5 post-mortem (Table S1, S2) (P<0.05).