Unveiling the Forensic Potential of Oral and Nasal Microbiota in Post-Mortem Interval Estimation

Microbiota have emerged as a promising tool for estimating the post-mortem interval (PMI) in forensic investigations. The role of oral and nasal microbiota in cadaver decomposition is crucial; however, their distribution across human cadavers at different PMIs remains underexplored. In this study, we collected 88 swab samples from the oral and nasal cavities of 10 healthy volunteers and 34 human cadavers. Using 16S rRNA gene sequencing, we conducted comprehensive analyses of the alpha diversity, beta diversity, and relative abundance distribution to characterize the microbial communities in both healthy individuals and cadavers at varying PMIs and under different freezing conditions. Random forest models identified <i>Firmicutes</i>, <i>Proteobacteria</i>, <i>Bacteroidota</i>, <i>Actinobacteriota</i>, and <i>Fusobacteriota</i> as potential PMI-associated biomarkers at the phylum level for both the oral and nasal groups, along with genus-level biomarkers specific to each group. These biomarkers exhibited nonlinear changes over increasing PMI, with turning points observed on days 5, 12, and 22. The random forest inference models demonstrated that oral biomarkers at both the genus and phylum levels achieved the lowest mean absolute error (MAE) values in the training dataset (MAE = 2.16 days) and the testing dataset (MAE = 5.14 days). Additionally, freezing had minimal impact on the overall phylum-level microbial composition, although it did affect the relative abundance of certain phyla. At the genus level, significant differences in microbial biomarkers were observed between frozen and unfrozen cadavers, with the oral group showing greater stability compared to the nasal group. These findings suggest that the influence of freezing should be considered when using genus-level microbial data to estimate PMIs. Overall, our results highlight the potential of oral and nasal microbiota as robust tools for PMI estimation and emphasize the need for further research to refine predictive models and explore the environmental factors shaping microbial dynamics.