Characterization of Bacterial Community Dynamics of the Human Mouth Throughout Decomposition via Metagenomic, Metatranscriptomic, and Culturing Techniques

Abstract

The postmortem microbiome has recently moved to the forefront of forensic research, and many studies have focused on the idea that predictable fluctuations in decomposer communities could be used as a “microbial clock” to determine time of death. Commonly, the oral microbiome has been evaluated using 16S rRNA gene sequencing to assess the changes in community composition throughout decomposition. We sampled the hard palates of three human donors over time to identify the prominent members of the microbiome. This study combined 16S rRNA sequencing with whole metagenomic (MetaG) and metatranscriptomic (MetaT) sequencing and culturing methodologies in an attempt to broaden current knowledge about how these postmortem microbiota change and might function throughout decomposition. In all four methods, Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the dominant phyla, but their distributions were insufficient in separating samples based on decomposition stage or time or by donor. Better resolution was observed at the level of genus, with fresher samples from decomposition clustering away from others via principal components analysis (PCA) of the sequencing data. Key genera in driving these trends included Rothia; Lysinibacillus, Lactobacillus, Staphylococcus, and other Firmicutes; and yeasts including Candida and Yarrowia. The majority of cultures (89%) matched to sequences obtained from at least one of the sequencing methods, while 11 cultures were found in the same samples using all three methods. These included Acinetobacter gerneri, Comamonas terrigena, Morganella morganii, Proteus vulgaris, Pseudomonas koreensis, Pseudomonas moraviensis, Raoutella terrigena, Stenotrophomonas maltophilia, Bacillus cereus, Kurthia zopfii, and Lactobacillus paracasei. MetaG and MetaT data also revealed many novel insects as likely visitors to the donors in this study, opening the door to investigating them as potential vectors of microorganisms during decomposition. The presence of cultures at specific time points in decomposition, including samples for which we have MetaT data, will yield future studies tying specific taxa to metabolic pathways involved in decomposition. Overall, we have shown that our 16S rRNA sequencing results from the human hard palate are consistent with other studies and have expanded on the range of taxa shown to be associated with human decomposition, including eukaryotes, based on additional sequencing technologies.

A conserved interdomain microbial network underpins cadaver decomposition despite environmental variables

“…Our network analysis identified multiple fungal members that are co-occurring with bacteria, belonging to the Ascomycota phylum (Fig. 5a )—a phylum known for its role in breaking down organic matter 6 , 44 , 52 , 53 . In particular, Yarrowia and Candida are known for their ability to utilize lipids, proteins and carbohydrates 44 , 53 , and both have one of their highest correlations with O. alkaliphila (Fig.…”

The central and biodynamic role of gut microbiota in critically ill patients

“…A more complete microbial composition can be assessed through unbiased sequencing of all DNA (shotgun metagenomics) present in a sample [ 23 ]. This higher resolution approach, although more expensive, allows the identification of bacteria up to species level and provides information on microbial richness, diversity and gene functions [ 23 , 24 , 26 ]. These approaches can be further informed by integrating them with proteins (metaproteomics) and small molecules (metabolomics) profiling.…”

“…This is a simple, fast and low-cost technique. Limitations include that it gives no information on gene functions and that two organisms with the same 16S rRNA gene sequence could be misclassified [23][24][25].…”

Toward Oral Thanatomicrobiology—An Overview of the Forensic Implications of Oral Microflora

“…The durability is influenced by the postmortem interval and other extrinsic factors. Though there is a significant change in the composition and abundance of the bacterial taxa over time, the overall diversity remains relatively stable and can be very helpful in the early postmortem period (12,(23)(24)(25)(26)(27)(28)(29)(30).…”