1. Ecology
  2. Microbiology and Infectious Disease
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A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system

  1. Jessica L Metcalf  Is a corresponding author
  2. Laura Wegener Parfrey
  3. Antonio Gonzalez
  4. Christian L Lauber
  5. Dan Knights
  6. Gail Ackermann
  7. Gregory C Humphrey
  8. Matthew J Gebert
  9. Will Van Treuren
  10. Donna Berg-Lyons
  11. Kyle Keepers
  12. Yan Guo
  13. James Bullard
  14. Noah Fierer
  15. David O Carter
  16. Rob Knight  Is a corresponding author
  1. University of Colorado at Boulder, United States
  2. University of Minnesota, United States
  3. Pacific Biosciences, United States
  4. Chaminade University of Honolulu, United States
  5. Howard Hughes Medical Institute, University of Colorado at Boulder, United States
Research Article
Cite this article as: eLife 2013;2:e01104 doi: 10.7554/eLife.01104
6 figures, 5 tables, 4 data sets and 1 additional file

Figures

We used two independent methods to assess stages of decomposition: (A) a visual body score estimate of decomposition following the Megyesi Key (Megyesi et al., 2005) and (B) the pH of soil to determine when rupture had occurred.

(A) visual key estimates for the head (orange circle): Fresh–no discoloration (1 point); Active Decay–Discoloration (2 points), Purging of decomposition fluids out of eyes, nose, or mouth (3 points), Bloating of neck and/or face (4 points); Advanced Decay–Sagging of flesh (5 points), Sinking of flesh (6 points), Caving in of flesh (7 points), Mummification (8 points). The key for the torso (blue triangle) is the same as above except that Bloating of abdominal cavity (3 points) precedes Rupture and/or purging of fluids (4 points). Gray boxes around points indicate generally with which stage of decay each time point is associated–Fresh (∼days 0, 3), Active Decay (∼days 6, 9, 13), and Advanced Decay (∼days 20, 34, 48). (B) Average pH of soil over time with standard error. A dramatic increase in pH occurred between day 6 and day 9, which is when rupture of body fluids and subsequent leakage into the soil likely occurred.

https://doi.org/10.7554/eLife.01104.004
Bacterial community composition changes significantly and consistently over the course of decomposition.

(A) Relative abundance of phyla of bacteria over time for all body sites. For the abdominal site, Day 0 includes cecum, fecal, and abdominal swab and liquid samples. For the soil site, control soils collected on days 0, 6, and 20 are shown on the left of the plot. (B) The three bacterial families that show the greatest change in abundance over time are plotted for each site. (C) PCoA plot based on unweighted UniFrac distances displaying bacterial community change at all sites during decomposition. Results from Mantel tests (Rho and p values) show that bacterial community change correlated significantly with time. The point of rupture is marked with a thick vertical black line on each plot.

https://doi.org/10.7554/eLife.01104.005
Eukaryotic community composition changes directionally and becomes dominated by the nematode Oscheius tipulae.

(A) PCoA plot based on unweighted UniFrac distances displaying microbial eukaryotic community change during decomposition. Results from Mantel tests (Rho and p values) show that microbial eukaryotic community change correlated significantly with time except for the skin of the belly. (B) Relative abundance of microbial eukaryote taxa at the class level over time. Microbial eukaryotic community composition changes significantly and predictably over the course of decomposition. (C) The eukaryotic nematode O. tipulae became highly abundant at each sample site at late stages of decomposition. The point of rupture is marked with a thick vertical black line on each plot.

https://doi.org/10.7554/eLife.01104.009
Phylogenetic distance (PD) alpha diversity and standard error for both bacterial (16S) and eukaryotic (18S) microbial communities at each sample site for Fresh vs Advanced Decay.

t test results are indicated by a single star for significant (p=0.01), two stars for highly significance (p<0.001), ‘MS’ for marginal significance (p∼0.05), and ‘NS’ for not significant. For soils, the Bonferoni corrected p value for a t test with three comparisons was 0.017. For bacterial communities, PD alpha diversity decreased at each sample site between the Fresh and Advanced Decay stages. For microbial eukaryotic communities, changes in PD alpha diversity were variable across sample sites—decreasing for soil, no significant change for the abdominal cavity, and increasing for skin. However, for Shannon diversity (evenness), microbial eukaryotic communities significantly decreased between the Fresh and Advanced Decay stages at every site (data not shown, soil p<0.001, abdominal p=0.002, skin p=0.03).

https://doi.org/10.7554/eLife.01104.011
Estimates of the mean absolute error (MAE) and standard deviations for PMI regressed directly on the taxon relative abundances (in days) based on bacterial (‘bac’) and eukaryotic (‘euk’) microbiome composition for each sampled site— soil (brown), abdominal cavity (teal), skin of the head (red), and skin of the body (pink).

Results are show for the timeframe of 0–34 days, which produced smallest errors. Results are displayed with the smallest MAE shown at the top (skin of head with combined 16S and 18S data) and largest error at the bottom (18S abdominal data).

https://doi.org/10.7554/eLife.01104.012
Trends of the postmortem interval (PMI) estimates.

(A) A regression of PMI predictions vs true PMI using 16S data. The line represents a perfect prediction of PMI. The abdominal cavity shows the greatest scatter of points deviating from the line. (B) Estimates of the mean absolute error (MAE) for PMI predictions using subsets of highly predictive bacterial taxa. Results suggest that 5–10 highly predictive taxa are required to recover MAEs similar to those generated using the entire microbial community. Similar results were discovered for 18S data, but results are not shown.

https://doi.org/10.7554/eLife.01104.013

Tables

Table 1
Total number of samples collected for each site and abdominal, skin (head and body), soil (with and without corpses)
https://doi.org/10.7554/eLife.01104.003
Sample typeSamples collectedSamples sequenced (16S HiSeq)Samples sequenced (18S Hiseq)Samples sequenced (PacBio)
Abdominal65432312
Skin of body5333310
Skin of head4036296
Soil with corpse5346608
Soil no corpse12980
Sum22316715226
  1. We show the number of successfully sequenced samples for each data type, including Illumina Hiseq and Pacific Biosciences. For Illumina data, we only included samples used in statistical analyses, which required>2500 sequences/sample. Details about each of the individual samples can be found in Supplementary file 1A.

Table 2
The five most changing bacterial taxa groups resolved to the family level for each site over the timeline of the experiment based on HiSeq Illumina results
https://doi.org/10.7554/eLife.01104.006
Rank changeSoilAbdominalSkin
1Gammaproteobacteria Xanthomonadales XanthomonadaceaeClostridia Clostridiales ClostridiaceaeEpsilonproteobacteria Campylobacterales Campylobacteraceae
2Alphaproteobacteria Caulobacterales CaulobacteraceaeBacilli Lactobacillales LactobacillaceaeGammaproteobacteria Pseudomonadales Pseudomonadaceae
3Sphingobacteria Sphingobacteriales SphingobacteriaceaeBacteroidia Bacteroidales BacteroidaceaeBetaproteobacteria Burkholderiales Alcaligenaceae
4Alphaproteobacteria Rhizobiales AurantimonadaceaeClostridia Clostridiales LachnospiraceaeSphingobacteria Sphingobacteriales Sphingobacteriaceae
5Alphaproteobacteria Sphingomonadales SphingomonadaceaeBacilli Lactobacillales EnterococcaceaeGammaproteobacteria Xanthomonadales Xanthomonadaceae
  1. Groups that increase in abundance are listed in bold text and groups that decreased in abundance are shown in normal text.

Table 3
For each sample site and each marker type, PERMANOVA results of UniFrac distance (unweighted) for Fresh (day 0–3 days) vs Advanced Decay (days 20–48) decomposition microbial communities
https://doi.org/10.7554/eLife.01104.007
PERMANOVA pseudo Fp value (999 permutations)
16S soil with corpse2.380.007
16S ctrl soil vs Advanced Decay soil2.540.001
16S abdominal6.310.001
16S skin on head8.190.001
16S skin on body5.810.001
18S soil with corpse10.170.001
18S ctrl soil vs Advanced Decay soil5.230.001
18S abdominal5.340.001
18S skin on head
18S skin on body5.960.001
  1. For soil sites, we also include comparisons of control soils vs Advanced Decay gravesoils. For the 18S skin of head, there were not sufficient samples for statistical analysis.

Table 4
For each sample site and each marker type, Mantel test results using Spearman’s rank correlation coefficient to assess the correlation between microbial community UniFrac distance (unweighted) and time
https://doi.org/10.7554/eLife.01104.008
Spearman RhoSpearman p value
16S soil0.5480.001
16S ctrl soil0.0510.352
16S abdominal0.3640.001
16S skin of head0.3680.001
16S skin of body0.4370.001
18S soil0.7720.001
18S ctrl soil0.1270.154
18S abdominal0.2090.029
18S skin of head0.2790.004
18S skin of body0.0790.143
  1. Importantly, control soil microbial communities did not change significantly over time.

Table 5
The five most changing microbial eukaryotes for each site over the timeline of the experiment based on HiSeq Illumina results
https://doi.org/10.7554/eLife.01104.010
Rank changeSoilAbdominalSkin
1Rhabditinae OscheiusRhabditinae OscheiusRhabditinae Oscheius
2Pythiaceae PythiumTritrichomonasRhizaria
3RhizariaZygosaccharomycesMucoraceae Rhizomucor
4AlveolataAscomycota GraphiumChromulinaceae Uroglena
5ZygomycetesNucleariidae FonticulaPythiaceae Pythium
  1. Groups that increase in abundance are listed in bold text and groups that decreased in abundance are shown in normal text.

Data availability

The following data sets were generated
  1. 1
  2. 2
  3. 3
    Metcalf_Mouse_Decomp_2011_18S_PacBio, QIIME Study ID 1997
    1. JL Metcalf
    2. LW Parfrey
    3. A Gonzalez
    4. CL Lauber
    5. D Knights
    6. G Ackermann
    et al. (2013)
    ID ERP003928. Publicly available at the EBI European Nucleotide Archive (http://www.ebi.ac.uk/ena/).
  4. 4
    Metcalf_Mouse_Decomp_2011_16S_PacBio, QIIME Study ID 1996
    1. JL Metcalf
    2. LW Parfrey
    3. A Gonzalez
    4. CL Lauber
    5. D Knights
    6. G Ackermann
    et al. (2013)
    ID ERP003929. Publicly available at the EBI European Nucleotide Archive (http://www.ebi.ac.uk/ena/).

Additional files

Supplementary file 1

(A) Excel file containing detailed metadata for each sample. (B) Excel file containing detailed OTU and taxonomy information for PacBio samples. (C) Excel file containing means, standard deviation, and two sample t test of Weighted Unifrac distances for Fresh versus Advanced Stage decomposition. (D) Excel file containing PMI estimates from the regression analyses and associated statistics. (E) Excel file containing taxa importance estimates associated with the PMI regression analyses.

https://doi.org/10.7554/eLife.01104.014

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