Estimating the time since deposition (TsD) in saliva stains using temporal changes in microbial markers

Determining the time since deposition (TsD) of traces could be helpful in the investigation of criminal offenses. However, there are no reliable markers and models available for the inference of short-term TsD. The goal of this study was to investigate the potential of the succession pattern of human salivary microbial communities to serve as an efficiency TsD prediction tool in the resolution of the forensic cases. Saliva stains exposed to indoor conditions up to 20 days were collected and analyzed by 16S rRNA profiling using high-throughput sequencing technique. Noticeable differences in microbial composition were observed between different time points, and the indoor exposure time of saliva stains were inversely correlated with alpha diversity estimates across the measured time period. The sequencing results were used to identify TsD-dependent bacterial indicators to regress a generalized random forest model, resulting in a mean absolute deviation (MAD) of 1.41 days. Furthermore, a simplified TsD predictive model was also developed utilizing Enhydrobacter, Paenisporosarcina, and Janthinobacterium by quantitative PCR (qPCR) with a MAD of 1.32 days, and then forensic practice assessment were also performed by using mock samples with a MAD of 3.53 days. In conclusion, this study revealed significant changes in salivary microbial abundance as the prolongation of TsD. It demonstrated that the microbial biomarkers could be invoked as a "clock" for TsD estimation in human dried saliva stains.