Impact of substrates and deposition time on bacteria associated with saliva samples and its implication on a bacterial signature-based body fluid identification method

Traditional serological methods for body fluid detection are limited by poor specificity and sensitivity, lacking confirmatory capability and thereby increasing the risk of false positives that may compromise the integrity of forensic investigations. While molecular methods such as mRNA, miRNA, and DNA methylation offer improved specificity, they are often hindered by marker instability and operational complexity in routine forensic workflows. Bacterial signature profiling has emerged as a promising alternative, offering enhanced stability, abundance, and compatibility with existing DNA workflows. However, its forensic implementation requires further validation. This study evaluated the effects of substrate type and storage time on the salivary microbiome to assess the reliability of its key bacterial signatures under conditions relevant to forensic casework. Saliva from ten donors was deposited onto six untreated substrates—including both fabric and non-fabric surfaces—and analyzed at four time points (Weeks 0, 1, 4, and 8) using 16S rDNA sequencing targeting the V4 region. Sequencing was performed on Illumina MiSeq FGx and NextSeq 2000 platforms, with downstream processing in Mothur v1.48.2 and data visualization in R and Excel. Results demonstrated that while bacterial community composition shifted significantly over time (e.g., succession from Firmicutes to increased Fusobacteria and Actinobacteria), substrate material had no significant impact on community structure. Core oral genera—including Veillonella, Streptococcus, and Prevotella—were consistently detected across all conditions, aligning with findings by Wohlfahrt et al. (2023). Post-hoc Tukey HSD analysis confirmed significant differences in alpha diversity between select time points, and NMDS analysis revealed sample clustering primarily by storage time rather than substrate type. Indicator species analysis showed no statistically significant genus-substrate associations, while several genera—including Veillonella and Leptotrichia—were significantly associated with specific time points, supporting the interpretation that salivary bacterial signatures are temporally dynamic yet remain stable across different surfaces. These findings underscore the forensic potential of bacterial profiling by demonstrating the temporal persistence and substrate-independent stability of key salivary taxa.