In the sometimes tricky business of establishing time of death, forensic investigators may have new allies—microbes. Bacterial and microbial eukaryotic organisms such as fungi, nematodes, and amoeba that teem over (and through) cadavers have tales to tell. To hear these tales, however, investigators must resort to high-technology gene sequencing techniques.
In cases in which the postmortem interval (PMI) in a mouse model system was actually 48 days, the researchers were able to consult a microbial clock—a sequence of postmortem microbial community changes—to give an estimate that was off by hardly more than three days.
This result was published in a paper entitled “A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system.” Appearing September 23 in eLIFE, an online science and biomedical journal, the paper notes that while establishing the time since death is critical in the initial stages of death investigations, existing techniques are susceptible to a range of errors and biases.
“Currently, investigators use tools ranging from the timing of last text messages and corpse temperatures to insect infestations on bodies and ‘grave soil’ analyses, with varying results,” said Jessica Metcalf, a postdoctoral researcher at the University of Colorado, Boulder and first author of the study. “Our results provide a detailed understanding of the bacterial changes that occur as mouse corpses decompose, and we believe this method has the potential to be a complementary forensic tool for estimating time of death.”
The researchers tracked microbial changes on the heads, torsos, body cavities, and associated grave soil of 40 mice at eight different time points over the 48-day study. The stages after death include the “fresh” stage before decomposition, followed by “active decay” that includes bloating and subsequent body cavity rupture, followed by “advanced decay.” At each stage, microbes may be reliably quantified using high-throughput DNA sequencing.
The researchers demonstrated that combining complementary sequencing technologies could yield high-resolution taxonomic data. These technologies included the Illumina HiSeq platform, which was used to sequence about 100 base pairs of both 16S and 18S amplicons at a depth of millions of sequences, and the Pacific Biosciences RS platform, which was used to sequence a lengthy fragment of the rRNA gene (roughly 800 base pairs for 16S amplicons and 1200 base pairs for 18S amplicons) at a level of thousands of sequences. Together, these techniques enabled the characterization of highly diverse microbial communities, as well as providing species and genus level taxonomic resolution of key taxa.
“At each time point that we sampled, we saw similar microbiome patterns on the individual mice and similar biochemical changes in the grave soil,” said Laura Parfrey, a former CU-Boulder postdoctoral fellow and now a faculty member at the University of British Columbia who is a microbial and eukaryotic expert. “And although there were dramatic changes in the abundance and distribution of bacteria over the course of the study, we saw a surprising amount of consistency between individual mice microbes between the time points—something we were hoping for.”
As part of the project, the researchers also charted “blooms” of a common soil-dwelling nematode well known for consuming bacterial biomass that occurred at roughly the same time on individual mice during the decay period. “The nematodes seem to be responding to increases in bacterial biomass during the early decomposition process, an interesting finding from a community ecology standpoint,” said Metcalf.
“This work shows that your microbiome is not just important while you’re alive,” said CU-Boulder Associate Professor Rob Knight, the corresponding study author who runs the lab where the experiments took place. “It might also be important after you’re dead.”