Species-level taxonomic characterization enhances the power of saliva and feces stain microbiota for inferring the time since deposition (TsD).

Accurate identification of body fluid stains found at crime scenes and inference of the time since deposition support forensic investigations. Forensic microbiology is recognized as a promising approach for addressing these challenges. Saliva and feces, which are frequently obtained as biological samples at crime scenes, contain greatly enriched bacterial communities. Therefore, we collected saliva and feces stains exposed to indoor environment. To simulate the time intervals typically encountered in forensic evidence examination, samples were exposed to indoor conditions for a 3-day short-term interval, and control samples were collected on the 3rd day to simulate the time point of arrest. We utilized the 16S rDNA full-length sequencing to characterize the microbial community. The results showed that microbial community composition of fresh body fluids, whether saliva or feces samples, remained stable on day 0 and day 3, with no substantial changes after 6 hour of exposure. The relative abundance of the dominant flora changed mainly after 24 h of exposure, such as an increase in Streptococcus oralis in saliva and a decrease in Bifidobacterium adolescentis in feces, and remained stable after 48 h and 72 h. The decrease in humidity seems to cause a reduction in some fluid flora, such as Prevotella melaninogenica in saliva. This suggests that collecting body fluid stain samples and recording environmental factors as early as possible after a case may increase the reliability of the evidence. In addition, saliva and feces stains exhibited distinct microbial composition profiles after exposure, indicating that they retained the potential for body fluid identification. The difference mainly stemmed from the respective dominant flora. Then, the time since deposition (TsD) prediction models were constructed, using a random forest regression algorithm, based on genus-level and species-level, respectively. The predictive efficacy of species-level microbial markers was higher, with the mean absolute error (MAE) of the saliva and feces prediction models being 0.30 day and 0.56 day, respectively. This finding suggests that using species-level microbial markers to determine the type and time since deposition (TsD) of body fluid stains has promising forensic value.