Successive bacterial colonisation of pork and its implications for forensic investigations

Aims: Bacteria are considered one of the major driving forces of the mammalian decomposition process and
have only recently been recognised as forensic tools. At this point, little is known about their potential use as
‘post-mortem clocks’. This study aimed to establish the proof of concept for using bacterial identification as
post-mortem interval (PMI) indicators, using a multi-omics approach.
Methods and Results: Pieces of pork were placed in the University’s outdoor facility and surface swabs were
taken at regular intervals up to 60 days. Terminal restriction fragment length polymorphism (T-RFLP) of the 16S
rDNA was used to identify bacterial taxa. It succeeded in detecting two out of three key contributors involved in
decomposition and represents the first study to reveal Vibrionaceae as abundant on decomposing pork.
However, a high fraction of present bacterial taxa could not be identified by T-RFLP. Proteomic analyses were
also performed at selected time points, and they partially succeeded in the identification of precise strains,
subspecies and species of bacteria that colonized the body after different PMIs.
Conclusion: T-RFLP is incapable of reliably and fully identifying bacterial taxa, whereas proteomics could help
in the identification of specific strains of bacteria. Nevertheless, microbial identification by next generation
sequencing might be used as PMI clock in future investigations and in conjunction with information provided by
forensic entomologists.
Significance and Impact of the Study: To the best of our knowledge, this work represents the first attempt to
find a cheaper and easily accessible, culture-independent alternative to high-throughput techniques to establish a
‘microbial clock’, in combination with proteomic strategies to address this issue.
Keywords: PMI; bacterial succession; T-RFLP; decomposition; microbiome; forensic science; metagenomics;
proteomics.