Visible decay progression
The decomposition process of the corpses of rats during 15 days was recorded and classified into five stages: The fresh stage began at 0.0 ± 0.0 h with no odor emitted; the bloat stage started along with body expansion giving off odorous gases at 2.6 ± 1.1 days; the active stage started at 5.0 ± 1.0 days with the body being ruptured by accumulated gases and several parts of the tissues were broken down along with plenty of liquid flowing out; the advanced stage began at 8.0 ± 1.0 days along with most parts of the tissues being removed; the dry stage happened at 12.5 ± 1.9 days with no soft tissue left.
Relative abundance of gut flora in different groups
A total of 7,029,815 raw and 6,674,323 clean reads were obtained by performing the high-throughput sequencing, with an effective rate of 94.97% (the ratio of clean to raw reads). A total of 22,625 OTUs were identified based on 97% similarity, 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 the samples were smooth as the number of sequences increased, demonstrating that this sequencing profundity could mirror the complete bacterial species richness among the samples (See Additional File 1). The basic information regarding the number of OTU and alpha diversity indices in individual rats revealed that these parameters in the samples collected at different time points after death reduced sharply in comparison to the living individuals (See Supplementary Table 1). A Venn diagram was plotted to compare the similarities and variances among the communities obtained in the different groups. The eleven groups showed communities of 36 OTUs in common, 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%, and 26.53% at time points corresponding to alive, 0 h, 8 h, 16 h, 1 day, 3 days, 5 days, 7 days, 9 days, 13 days, and 15 days, respectively, (Fig. 1). Ternary plots were constructed using the Ternary plot order of the VCD package in R software. In Fig. 2a, we observed that Corynebacterium amycolatum exhibited the highest abundance and the highest load at 0 h time point among the three-time points and that Bacterium mpn isolate group 2 and Falsiporphyromonas endometrii were the highest in living individuals and at 8 h after death time point, respectively. In Fig. 2b, we observed that the richness of Enterococcus faecalis increased at 16 h time point, and at 1 and 3 days postmortem, followed by Proteus mirabilis, which was highest at 3 days postmortem as compared to the other two time points. In Fig. 2c, we observed that the relative abundance of E. faecalis increased at 5 days postmortem, and exhibited almost the same abundance at 7 and 9 days postmortem. In Fig. 2d, we observed that P. mirabilis exhibited the highest abundance among all the three-time points on day 15 after death, followed by Vagococcus lutrae and E. faecalis that were much higher at 9 and 13 days postmortem than that at 15 days after death, respectively.
Microbial analysis at different levels
The microbial community structure was determined during the succession of decomposition, and all the 16S rRNA sequences were classified at the phylum, genus, and species levels. The notable tendencies and fluctuations exhibited the relative richness of the diverse bacterial taxa in the rectum of the rat cadavers through the decaying process (Fig. 3a, b, c; Fig. 4a, b, c; Table 1). Figures 3a, b, c showed the variations in the proportions of bacteria at different levels, and Figs. 4a, b, c showed the relative abundance of the ten topmost bacteria identified in the study samples.
Table 1
Significant difference in Abundance of OTUs at phylum, genera and species levels in pre- and post-mortem
Bacteria
|
Mean relative abundance
|
alive
|
h0
|
h8
|
h16
|
D1
|
D3
|
D5
|
D7
|
D9
|
D13
|
D15
|
Phylum
|
|
|
|
|
|
|
|
|
|
|
|
Proteobacteria
|
0.018967a
|
0.238017c
|
0.685157b
|
0.515944
|
0.783757bd
|
0.603994b
|
0.477884
|
0.509146
|
0.655225b
|
0.445179
|
0.546184
|
Bacteroidetes
|
0.575744a
|
0.055408
|
0.046913b
|
0.039948
|
0.009058
|
0.090197
|
0.002348b
|
0.003179b
|
0.000968b
|
0.001218b
|
0.055198b
|
Actinobacteria
|
0.00136
|
0.176496c
|
0.008813
|
0.011762 g
|
0.002587
|
0.00605
|
0.000186dh
|
0.000137dh
|
0.000298d
|
0.001174d
|
0.000166dh
|
Genera
|
|
|
|
|
|
|
|
|
|
|
|
Enterococcus
|
0.000381a
|
0.127666
|
0.013376e
|
0.016927 g
|
0.028338i
|
0.207753b
|
0.491857bfhj
|
0.382742b
|
0.332367b
|
0.374643bj
|
0.217481b
|
Proteus
|
0.000049a
|
0.00023c
|
0.000577e
|
0.000455 g
|
0.000597i
|
0.104454bdfj
|
0.044976bfj
|
0.046492
|
0.084841
|
0.216454bdfhj
|
0.466327bdfhj
|
Lactobacillus
|
0.022459a
|
0.189569c
|
0.174197e
|
0.336959 g
|
0.162977i
|
0.015514
|
0.003732
|
0.045177h
|
0.001712h
|
0.000176bdfhj
|
0.000616dfh
|
Vagococcus
|
0.00001a
|
0.00047
|
0.003551
|
0.004299
|
0.003737
|
0.03471b
|
0.005624b
|
0.014966b
|
0.00558b
|
0.045661b
|
0.120857b
|
Helicobacter
|
0.001624a
|
0.041934c
|
0.000572
|
0.00201 g
|
0.000059
|
0bdh
|
0bdh
|
0bdh
|
0bdh
|
0.000005bd
|
0.000044bd
|
Species
|
|
|
|
|
|
|
|
|
|
|
|
Enterococcus_faecalis
|
0.000059a
|
0.008583
|
0.001355e
|
0.006862
|
0.01947i
|
0.115879b
|
0.39001bfj
|
0.241289bf
|
0.230305b
|
0.22268bfj
|
0.103055b
|
Proteus_mirabilis
|
0.000034a
|
0.000127c
|
0.000372e
|
0.000372 g
|
0.00045i
|
0.079285bdfj
|
0.026675bdfj
|
0.036319
|
0.071259
|
0.180374bdfhj
|
0.281585bdfhj
|
Clostridium_sporogenes
|
0a
|
0c
|
0.000352e
|
0g
|
0i
|
0.000024
|
0.000044
|
0.00021
|
0.000386
|
0.104938bdfhj
|
0.027144bdfhj
|
Corynebacterium_amycolatum
|
0.000044
|
0.000665c
|
0.001585e
|
0.001047 g
|
0.000181
|
0.008432dfh
|
0.014164dfh
|
0.043347dfh
|
0.001477dfh
|
0.00114dfh
|
0.006735dfh
|
Lactobacillus_intestinalis
|
0.00045
|
0.012618c
|
0.0206
|
0.027188 g
|
0.019549
|
0.000298
|
0.000034dh
|
0.000088dh
|
0.000015dh
|
0dh
|
0.000054dh
|
bacterium_mpn-isolate_group_2
|
0.025628a
|
0.000044
|
0.000034b
|
0.000015b
|
0.000015b
|
0.000005b
|
0b
|
0b
|
0b
|
0b
|
0.000005b
|
Lactobacillus_reuteri
|
0.006877a
|
0.014628c
|
0.00941
|
0.018869 g
|
0.017822
|
0.006383
|
0.000191
|
0.002005
|
0.000127
|
0.000015bdh
|
0.000196bdh
|
The significant findings were testified with Kruskal-Wallis test for Dunn's multiple comparison test and P<0.05 by GraphPad Prism. “a, c, e, g, i” represent alive, h0, h8, h16 and D1 pre- and post-mortem comparing with other time points, and “b, d, f, h, j” means having significant difference (P<0.05).
At the phylum level, Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria were found to be present at all the time points. Bacteroidetes (54.57%), Firmicutes (45.83%), and Proteobacteria were the dominant phylum in the living samples at 0 h postmortem and at other time points. Bacteroidetes began to show a declining trend and disappeared eventually, increasing again to 5.52% in the 15-day postmortem samples. The relative abundance of Proteobacteria in the living samples was much lower than that at 8 h, 1 day, 3 days, and 9 days postmortems, while the relative abundance of Bacteroidetes was much higher in the living samples than that at 8 h, 5 days, 7 days, 9 days, 13 days, and 15 days postmortem, and the relative abundance of Actinobacteria was significantly higher in 0 h than that at days 5, 7, 9, 13, and 15 postmortems (P < 0.05).
At phylum level, there was obvious change in bacterial richness following body decomposition, which declined by 52.38% from 0 h to day 9 (216 h) first and then increased by 49.95% from day 9 to day 15 (360 h) (y = –0.0737x + 0.0002x2 + 12.5770, R2 = 0.390, P = 5.474e-09) (Figure 5a).
At the genus level, Lactobacillus and Enterococcus appeared as the dominant genera at day 1 and 3–13 days postmortem, respectively, Helicobacter disappeared at days 7, 9, and 15 PMIs and Proteus was the most abundant at day 15 of postmortem. The relative abundance of rectum flora in the living samples was significantly lower than that at days 3, 5, 7, 9, and 13 PMIs; nevertheless, Helicobacter was much higher in the living samples than those in the postmortems of the above-mentioned days (P < 0.05). The proportion of Proteus was significantly lower in the living samples and the 0 h, 8 h, 16 h, and day 1 postmortem samples than those in the 13- and 15-day postmortem samples, while Lactobacillus exhibited an opposite result (P < 0.05).
At the genus level, there was significant shift in bacterial richness during 0 h to day 15 decomposition process (Figure 5b). Genus richness presented downward (78.36%) and upward (66.64%) trends, whereas the lowest time point turned out at day 9 (y =–0.8260x + 0.0020x2 + 108.6, R2 = 0.384, P = 7.881e-09).
At the species level, among the ten topmost species that existed at 8 h postmortem, Clostridium sporogenes and F. endometrii disappeared before 1-day postmortem and after 3 days postmortem, respectively. E. faecalis and P. mirabilis appeared during the whole decomposition process of 15 days after death; however, the former showed a downward trend from day 5 of postmortem, while the latter showed an upward trend. Bacterium mpn isolate group 2 disappeared during 5–13 days postmortem or decreased after death. The relative abundance of E. faecalis at days 3, 5, 7, 9, 13, 13, and 15 after death was much higher than that in the living samples, while Bacterium mpn isolate group 2 was distinctly lower (P < 0.05). The relative abundance of C. amycolatum was markedly lower in 0 h, 8 h, 16 h than in 7, 9, 13, and 15 days postmortem samples, while Lactobacillus reuteri and L. intestinalis were higher in 0 h, 16 h than at 13- and 15-days postmortem samples.
The species taxon richness first decreased (81.27%) then increased (57.01%) with the decomposition process and the turning point appeared at day 9 (y =–0.4382x + 0.0010x2 + 62.2575, R2 = 0.339, P = 1.214e-07) (Fig. 5c).
Characterization of bacterial diversity and community structure
The complete rectal flora community was evaluated based on diversity and richness, which was calculated at 97% similarity. Alpha diversity indices of the observed species, abundance-based coverage estimators (ACE), and chao1 values for the rectal bacteria in the living samples were significantly higher than those in the 5, 7, 9, 13, and 15 days postmortem samples, suggesting that the richness and diversity of the rectal flora declined significantly after day 5 of postmortem compared with the alive group (Table 2). The richness indices (ACE and chao1) went up slightly, however, there was no significant difference compared with other time points. All the alpha diversity indices are presented in Table 2, and there were significant differences in the overall rectal bacterial community structure among the eleven postmortem intervals.
The similarities in the gut flora communities of rats among the eleven groups were estimated using the beta diversity metrics, such as NMDS and beta diversity heatmap. As presented in Fig. 6a, the differences in coefficients among all the groups were almost higher than 0.5, indicating that the bacterial community in different groups exhibited great diversity. All the samples were clustered into 11 prime clusters. According to the NMDS (stress = 0.152), the bacterial communities of the gut samples were separated into three clusters between the late and early PMI (Fig. 6b). Conspicuously, the 8-h postmortem interval could be significantly separated from the other groups, indicating that the gut flora at 8 h after death differed from the other two clusters.
LEfSe is a biomarker detection and descriptive tool for performing high-dimensional statistics. The LEfSe analysis was performed to compare the projected bacterial community among the 11-time intervals at different levels (Fig. 6c). The results of this analysis suggested that the provision of related taxa was significantly diverse among all the groups. The LDA scores revealed that the relative abundances of C. amycolatum, Entero isolate group 2, Bacteroides uniformis, E. faecalis, Streptococcus gallolyticus subsp macedonics, and C. sporogenes were most abundant at postmortem intervals of 0 h, 1 day, 3 days, 5 days, 7 days, and 13 days, respectively, while P. mirabilis and V. lutrae were most abundant on day 15 of postmortem.
Table 2
Alpha diversity index of high-throughput analysis of intestinal microbial richness and diversity
Time points
|
Observed_species
|
Shannon
|
Simpson
|
Chao1
|
ACE
|
Goods_coverage
|
PD_whole_tree
|
alive
|
574a
|
6.284a
|
0.957a
|
681.781a
|
691.263a
|
0.995a
|
47.217a
|
h0
|
410c
|
4.845
|
0.842
|
472.923c
|
474.917c
|
0.997
|
45.098
|
h8
|
191
|
2.77b
|
0.681b
|
256.809
|
276.089
|
0.998
|
29.156
|
h16
|
321e
|
3.67
|
0.772
|
393.452e
|
414.277e
|
0.997e
|
54.33e
|
D1
|
159
|
2.414b
|
0.665b
|
238.27
|
269.216
|
0.997
|
23.572
|
D3
|
167
|
3.316
|
0.781
|
210.444
|
214.123
|
0.998
|
20.155
|
D5
|
61bdf
|
2.437
|
0.658b
|
80.908bdf
|
92.725bdf
|
0.999bf
|
14.241bf
|
D7
|
64bd
|
2.633b
|
0.739
|
78.199bdf
|
90.613bdf
|
0.999bf
|
10.862bf
|
D9
|
81bd
|
2.629b
|
0.705
|
107.505bd
|
114.157bd
|
0.999bf
|
13.614bf
|
D13
|
102b
|
2.388b
|
0.656b
|
148.395b
|
163.889b
|
0.998b
|
17.613b
|
D15
|
103bd
|
2.60b
|
0.697b
|
134.112bd
|
139.958bd
|
0.999bf
|
11.867bf
|
P*
|
< 0.0001
|
< 0.0001
|
0.0011
|
< 0.0001
|
< 0.0001
|
< 0.0001
|
< 0.0001
|
The values signified in the table are the mean values of each group, significant findings were testified with Kruskal-Wallis test for Dunn's multiple comparison test and P < 0.05 by GraphPad Prism. “a, c, e” represents alive, h0, h16 pre-and post-mortem comparing with other time points, and “b, d, f” means having significant difference.
Constructing a model for PMI
The best subset selection was combined with phylum, genus, and species indicators to construct the model that best explained aberrance in time of death from 0 h to day 15 (See Additional file 2 and 3; Table 3). Table 3 showed that the poorest model for PMI appeared at the phylum level (16.1% of the variation). The taxon that most contributed to postmortem existed at the genus level. The best subset selection results showed that seven bacteria at the genus level were selected as the best features to develop the model. These seven genera were: Enterococcus, Proteus, Lactobacillus, unidentified Clostridiales, Vagococcus, unidentified Corynebacteriaceae, and unidentified Enterobacteriaceae, and this model contained the most information and explained 87.2% (generalized cross-validation score (GCV) = 0.307) variation of time of death in this study. The species, including four bacteria, were identified from best subset selection as the most informative and explained 56.6% (GCV = 0.515). This model was poorer in explaining the variation in time of death than the model constructed by the genus features.
Table 3
Generalized additive models estimating death time utilizing taxonomic phyla, genus and species level indicators from Best Subset Features
Model
|
PMI (h) =
|
Percent (%)
|
R2(adj.)
|
GCV
|
|
Phylum
|
|
|
|
1
|
s(Proteobacteria) + s(Firmicutes) + s(Bacteroidetes) + s(Actinobacteria) + s(unidentified_Bacteria) + s(Spirochaetes) + s(Oxyphotobacteria) + s(Melainabacteria) + s(Fusobacteria) + s(Tenericutes)
|
45.0
|
0.344
|
0.792
|
2
|
s(Proteobacteria) + s(Firmicutes) + s(Bacteroidetes) + s(Actinobacteria) + s(unidentified_Bacteria) + s(Spirochaetes) + s(Oxyphotobacteria) + s (Melainabacteria) + s(Fusobacteria)
|
45.0
|
0.354
|
0.769
|
3
|
s(Proteobacteria) + s(Firmicutes) + s(Bacteroidetes) + s(Actinobacteria) + s(unidentified_Bacteria) + s(Oxyphotobacteria) + s (Melainabacteria) + s(Fusobacteria)
|
41.7
|
0.325
|
0.791
|
4
|
s(Proteobacteria) + s(Firmicutes) + s(Bacteroidetes) + s(Actinobacteria) + s(unidentified_Bacteria) + s(Oxyphotobacteria) + s(Melainabacteria)
|
39.8
|
0.313
|
0.793
|
5
|
s(Proteobacteria) + s(Firmicutes) + s(Bacteroidetes) + s(Actinobacteria) + s(unidentified_Bacteria) + s(Melainabacteria)
|
39.8
|
0.322
|
0.773
|
6
|
s(Proteobacteria) + s(Firmicutes) + s(Bacteroidetes) + s(unidentified_Bacteria) + s(Melainabacteria)
|
39.8
|
0.331
|
0.752
|
7
|
s(Proteobacteria) + s(Firmicutes) + s(Bacteroidetes) + s(Melainabacteria)
|
39.5
|
0.337
|
0.735
|
8
|
s(Proteobacteria) + s(Firmicutes) + s(Melainabacteria)
|
37.7
|
0.327
|
0.735
|
9
|
s(Proteobacteria) + s(Firmicutes)
|
24.1
|
0.194
|
0.867
|
10
|
s(Firmicutes)
|
16.1
|
0.123
|
0.930
|
|
Genus
|
|
|
|
11
|
s(Enterococcus) + s(Proteus) + s(Lactobacillus) + s(unidentified_Clostridiales) + s(Vagococcus) + s(unidentified_Corynebacteriaceae) + s(unidentified_Enterobacteriaceae) + s(Bacteroides)
|
85.3
|
0.792
|
0.307
|
12
|
s(Enterococcus) + s(Proteus) + s(Lactobacillus) + s(unidentified_Clostridiales) + s(Vagococcus) + s(unidentified_Corynebacteriaceae) + s(unidentified_Enterobacteriaceae)
|
87.2
|
0.803
|
0.307
|
13
|
s(Enterococcus) + s(Proteus) + s(unidentified_Clostridiales) + s(Vagococcus) + s(unidentified_Corynebacteriaceae) + s(unidentified_Enterobacteriaceae)
|
81.4
|
0.754
|
0.330
|
14
|
s(Enterococcus) + s(Proteus) + s(unidentified_Clostridiales) + s(Vagococcus) + s(unidentified_Enterobacteriaceae)
|
76.1
|
0.702
|
0.377
|
15
|
s(Enterococcus) + s(Proteus) + s(unidentified_Clostridiales) + s(unidentified_Enterobacteriaceae)
|
67.7
|
0.634
|
0.421
|
16
|
s(Enterococcus) + s(Proteus) + s(unidentified_Clostridiales)
|
61.6
|
0.572
|
0.484
|
17
|
s(Enterococcus) + s(Proteus)
|
44.9
|
0.434
|
0.588
|
18
|
s(Proteus)
|
37.4
|
0.338
|
0.708
|
|
Species
|
|
|
|
19
|
s(Enterococcus_faecalis) + s(Proteus_mirabilis) + s(Clostridium_sporogenes) + s(Vagococcus_lutrae) + s(Corynebacterium_amycolatum) + s(Streptococcus_gallolyticus_subsp_macedonicus) + s(Lactobacillus_intestinalis) + s(bacterium_mpn_isolate_group_2) + s(Lactobacillus_reuteri) + s(Falsiporphyromonas_endometrii)
|
59.8
|
0.528
|
0.561
|
20
|
s(Enterococcus_faecalis) + s(Proteus_mirabilis) + s(Clostridium_sporogenes) + s(Vagococcus_lutrae) + s(Corynebacterium_amycolatum) + s(Streptococcus_gallolyticus_subsp_macedonicus) + s(Lactobacillus_intestinalis) + s(bacterium_mpn_isolate_group_2) + s(Lactobacillus_reuteri)
|
59.8
|
0.535
|
0.545
|
21
|
s(Enterococcus_faecalis) + s(Proteus_mirabilis) + s(Clostridium_sporogenes) + s(Vagococcus_lutrae) + s(Corynebacterium_amycolatum) + s(Lactobacillus_intestinalis) + s(bacterium_mpn_isolate_group_2) + s(Lactobacillus_reuteri)
|
59.8
|
0.541
|
0.530
|
22
|
s(Enterococcus_faecalis) + s(Proteus_mirabilis) + s(Clostridium_sporogenes) + s(Vagococcus_lutrae) + s(Corynebacterium_amycolatum) + s(Lactobacillus_intestinalis) + s(bacterium_mpn_isolate_group_2)
|
59.5
|
0.545
|
0.518
|
23
|
s(Enterococcus_faecalis) + s(Proteus_mirabilis) + s(Clostridium_sporogenes) + s(Vagococcus_lutrae) + s(Lactobacillus_intestinalis) + s(bacterium_mpn_isolate_group_2)
|
59.1
|
0.546
|
0.510
|
24
|
s(Enterococcus_faecalis) + s(Proteus_mirabilis) + s(Clostridium_sporogenes) + s(Vagococcus_lutrae) + s(Lactobacillus_intestinalis)
|
58.1
|
0.54
|
0.511
|
25
|
s(Enterococcus_faecalis) + s(Proteus_mirabilis) + s(Clostridium_sporogenes) + s(Vagococcus_lutrae)
|
56.6
|
0.53
|
0.515
|
26
|
s(Enterococcus_faecalis) + s(Proteus_mirabilis) + s(Vagococcus_lutrae)
|
51.2
|
0.477
|
0.568
|
27
|
s(Enterococcus_faecalis) + s(Proteus_mirabilis)
|
49.4
|
0.432
|
0.646
|
28
|
s(Proteus_mirabilis)
|
25.5
|
0.246
|
0.773
|
The whole models were evaluated by the adjusted R2 value and the generalized cross-validation score (GCV). A higher R2 and lower GCV suggest better model. The percent variation of PMI (%) explained by per model. |
PICRUSt
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 (See Additional file 4 and 5). The top four different pathways at the Kyoto Encyclopedia of Genes and Genomes (KEGG) at levels 1 and 2 have been shown in Additional file 4, 5 and Table 4, 5. Among them, the functional pathways associated with metabolism, including carbohydrate and amino acid metabolisms, corresponded to a large number of related genes in all the samples (Table 4 and 5). The pathways related to environmental information processing and organismal systems were significantly higher in the rectal bacterial community on days 5, 7, 9, 13, and 15 after death as compared to the living samples (Table 4 and 5). Amino acid metabolism, energy metabolism, and metabolism of cofactors and vitamin pathways were significantly different between the living and 5, 7, and 9 days postmortem samples, while energy metabolism showed notable differences between the living and 5 days postmortem samples (Table 4 and 5) (P < 0.05). Although the pathway of carbohydrate metabolism did not differ significantly between the living and postmortem samples, the relative abundance increased in the 0 h and 3 days postmortem samples compared to that in the living sample.
Table 4 The relative abundance of KEGG level 1 and its significant difference among groups
KO_Hierarchy
|
Metabolism
|
Genetic_Information_Processing
|
Environmental_Information_Processing
|
Cellular_Processes
|
Human_Diseases
|
Organismal_Systems
|
alive
|
48.11±1.32a
|
20.89±0.91a
|
12.13±1.84a
|
3.41±0.57
|
0.74±0.04a
|
0.77±0.05a
|
h0
|
47.10±1.78c
|
19.08±2.37
|
15.24±1.68c
|
2.79±0.92
|
0.93±0.15
|
0.63±0.14c
|
h8
|
44.32±2.03e
|
17.49±1.47
|
16.39±1.62e
|
3.40±0.51
|
1.06±0.12b
|
0.49±0.10
|
h16
|
45.1±1.75
|
18.27±1.54
|
15.95±0.99
|
3.12±0.51
|
1.03±0.10
|
0.50±0.09
|
D1
|
43.5±0.80b
|
17.17±1.22b
|
17.06±0.56
|
3.51±0.46
|
1.11±0.08b
|
0.45±0.01
|
D3
|
44.25±0.80
|
17.44±1.27
|
17.21±1.71
|
3.22±0.60
|
1.03±0.13
|
0.47±0.07
|
D 5
|
42.78±0.30bdf
|
18.70±1.91
|
18.67±0.70bdf
|
2.71±0.81
|
0.98±0.11
|
0.36±0.08bd
|
D 7
|
43.38±0.58bd
|
18.28±1.67
|
18.38±0.97bd
|
2.86±0.71
|
0.99±0.13
|
0.39±0.05bd
|
D 9
|
42.92±0.37bd
|
17.73±1.84
|
18.23±0.78bd
|
3.16±0.80
|
1.05±0.13
|
0.40±0.07bd
|
D 13
|
43.39±0.50
|
17.70±1.44
|
18.33±1.39bd
|
3.44±1.05
|
0.99±0.26
|
0.40±0.05bd
|
D15
|
43.80±1.44
|
17.76±0.88
|
17.25±2.41b
|
3.52±0.61
|
1.12±0.25b
|
0.43±0.01b
|
The values signified in the table are the mean±SD of each time points, significant findings were testified with Kruskal-Wallis test for Dunn's multiple comparison test and P<0.05 by GraphPad Prism. “a, c, e” represents alive, h0, h8 pre-and post-mortem comparing with other time points, and “b, d, f” means having significant difference.
Table 5 The relative abundance of KEGG level 2 and their significant difference among groups
KO_Hierarchy
|
Membrane_Transport
|
Amino_Acid_Metabolism
|
Replication_and_Repair
|
Energy_Metabolism
|
Translation
|
Metabolism_of_Cofactors_and_Vitamins
|
Nucleotide_Metabolism
|
alive
|
10.45±1.65a
|
9.66±0.25a
|
9.55±0.53a
|
6.04±0.31a
|
6.10±0.35a
|
4.41±0.23a
|
4.36±0.24a
|
h0
|
13.21±1.59c
|
9.11±1.00c
|
8.52±1.21
|
5.47±0.50c
|
5.50±0.96
|
4.01±0.48
|
4.02±0.64
|
h8
|
13.80±1.23e
|
8.31±0.71
|
7.73±0.76
|
5.12±0.29
|
4.57±0.71
|
4.03±0.31
|
3.67±0.38
|
h16
|
13.58±0.75
|
8.27±0.70
|
8.13±0.79
|
5.14±0.20
|
4.97±0.70
|
3.81±0.24
|
3.87±0.40
|
D1
|
14.33±0.30
|
8.02±0.15
|
7.57±0.64b
|
5.03±0.03
|
4.42±0.58b
|
3.96±0.20
|
3.59±0.33b
|
D3
|
14.72±1.85
|
8.07±0.51
|
7.71±0.75
|
5.08±0.19
|
4.62±0.64
|
3.74±0.53
|
3.58±0.15
|
D 5
|
16.30±1.08bdf
|
7.25±0.64bd
|
8.46±1.08
|
4.79±0.15bd
|
5.28±0.97
|
3.27±0.53b
|
3.63±0.21
|
D 7
|
16.00±1.17b
|
7.58±0.50bd
|
8.19±0.93
|
4.84±0.12bd
|
5.08±0.83
|
3.36±0.49b
|
3.67±0.28
|
D 9
|
15.68±1.08b
|
7.61±0.68bd
|
7.90±1.06
|
4.87±0.15bd
|
4.79±0.93
|
3.55±0.50b
|
3.52±0.19b
|
D 13
|
15.86±1.62b
|
7.91±0.74
|
7.79±0.90
|
4.91±0.16b
|
4.86±0.74
|
3.44±0.51b
|
3.57±0.18b
|
D15
|
14.84±2.44b
|
8.06±0.45
|
7.75±0.55
|
5.03±0.32b
|
4.80±0.41
|
3.67±0.61
|
3.77±0.22
|
The values signified in the table are the mean±SD of each time points, significant findings were testified with Kruskal-Wallis test for Dunn's multiple comparison test and P<0.05 by GraphPad Prism. “a, c, e” represents alive, h0, h8 pre-and post-mortem comparing with other time points, and “b, d, f” means having significant difference.